Cell type–specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP

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978 | VOL.7 NO.5 | 2012 | nature protocols

IntroDuctIonMulticellularity is an inherent property of higher eukaryotes, and it drives complexity in terms of tissue diversity and function, as well as in terms of organismal diversity and evolution. The genera-tion of diverse cell types during embryonic development occurs through the regulation of precise spatial and temporal programs of gene expression. High-resolution in situ hybridization1–3 and single-cell RNA-seq methods4 are beginning to uncover these com-plex gene expression signatures for different cell types and between cell populations of a given tissue. However, our understanding of how these programs of gene expression are generated has remained a much more challenging issue to address. Cell culture–based systems have been extremely useful for uncovering basic princi-ples of transcriptional regulation; however, cells grown in culture are a poor proxy for in vivo tissue development, as they lack their developmental context, including signaling events arising from neighboring, interacting tissues.

ChIP analyses of transcription factor (TF) occupancy focusing on tissue-specific factors within the context of a developing embryo have proved to be very successful in dissecting regulatory programs driving particular cell fates and tissue development5–10. However, most TFs are used in many tissues and have pleiotropic roles in development. Similarly, transcriptional cofactors (for example, Groucho, CBP) and chromatin remodeling factors have very broad or ubiquitous expression throughout the embryo. Analysis of regulatory proteins and chromatin modifications from whole embryos therefore generates mixed and diluted signatures reflect-ing heterogeneous cell types and obscures dynamic changes in cases in which a gene is switching off in one tissue and switching on in another. Interpreting whole embryo TF occupancy data for broadly expressed factors is also challenging, as it is not possible to discern whether a TF occupies a given enhancer in all tissues in which it is expressed or only in a subset. These merged signals thereby make it difficult to disentangle regulatory connections and impede our understanding of how cell type diversity is generated during embryonic development.

Substantial progress has been made in addressing this crucial bottleneck in plants. FACS has been used to examine cell type–specific RNA expression signatures by sorting protoplasts11 or nuclei12.

A more broadly applicable approach, to both RNA signatures and ChIP, is based on the isolation of nuclei tagged in specific cell types (INTACT), which was used to study differences in chroma-tin modification between two cell types in Arabidopsis13. INTACT relies on biochemical affinity-based purification of tagged nuclei, based on placing a tag (BLRP) on a nuclear pore complex compo-nent, which is cell type–specifically biotinylated in the presence of a BLRP-specific biotin ligase (BirA). The method has high purity and good recovery making it a very promising approach for use in other model systems, exemplified by its recent application to unfixed Drosophila embryos14.

Obtaining cell type–specific information on transcriptional regulation during metazoan development remains a key challenge. The two commonly used methods involve either tissue dissection or FACS. Tissue dissection has been successfully used to examine p300 occupancy15,16 and chromatin signatures17,18 during mouse development, providing a systematic view of tissue-specific signa-tures during development. It is difficult, however, to move from a tissue-level view to cell type–specific signatures. Most tissues con-tain heterogeneous cell types and, in many cases, their accurate dissection is not possible as the population of cells is too small or interconnected with other structures. Often dissection is only possible after a tissue has formed, precluding an analysis of the progression of cell specification. Laser capture–based methods have improved the ability to dissect smaller regions19, but they are extremely time consuming and effectively not yet scalable. Dissection methods can also introduce potential inconsistencies as a result of human error.

FACS of cells or nuclei generated from dissociated embryos or tissues has been used to examine cell type–specific RNA expres-sion signatures20 and for native, unfixed ChIP21. Although native ChIP-seq can provide a comprehensive view of nucleosome posi-tioning22 and chromatin modifications23, an analysis of chromatin-binding proteins, including TFs, cofactors and chromatin remodelers, is not possible as the majority of these associations are transient and are readily lost during chromatin preparation and sonication before immunoprecipitation. Covalent cross-linking of embryos before dissociation avoids this issue by preserving

Cell type–specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIPStefan Bonn, Robert P Zinzen, Alexis Perez-Gonzalez, Andrew Riddell, Anne-Claude Gavin & Eileen E M Furlong

Genome Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany. Correspondence should be addressed to E.E.M.F. ([email protected]).

Published online 26 April 2012; doi:10.1038/nprot.2012.049

this protocol describes the batch isolation of tissue-specific chromatin for immunoprecipitation (Bits-chIp) for analysis of histone modifications, transcription factor binding, or polymerase occupancy within the context of a multicellular organism or tissue. embryos expressing a cell type–specific nuclear marker are formaldehyde cross-linked and then subjected to dissociation. Fixed nuclei are isolated and sorted using Facs on the basis of the cell type–specific nuclear marker. tissue-specific chromatin is extracted, sheared by sonication and used for chIp-seq or other analyses. the key advantages of this method are the covalent cross-linking before embryo dissociation, which preserves the transcriptional context, and the use of Facs of nuclei, yielding very high purity. the protocol has been optimized for Drosophila, but with minor modifications should be applicable to any model system. the full protocol, including sorting, immunoprecipitation and generation of sequencing libraries, can be completed within 5 d.

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nature protocols | VOL.7 NO.5 | 2012 | 979

DNA-protein interactions for unstable complexes. However, cross-linking generates a different problem—the inability to dis-sociate fixed embryos into single intact cells that can be accurately FACS sorted. To circumvent this issue, embryo dissociation can be performed before formaldehyde fixation of cells24; however, this approach runs the risk of introducing an aberrant stress response and nonphysiological changes to the transcriptional and chromatin context.

Here we describe a method that combines the advantages of embryo fixation with the purity and ease of FACS, allowing for the batch isolation of cell type– and tissue-specific chromatin from developing metazoan embryos for subsequent ChIP and down-stream analyses (deep sequencing or microarray; Fig. 1). The method requires a nuclear protein (we used an epitope-tagged histone H2B) that is exclusively expressed in the cell type of inter-est, which can be fluorescently labeled. BiTS-ChIP is based on the covalent cross-linking of transgenic embryos, from which intact fixed nuclei are isolated. FACS is then used to sort fluorescently labeled nuclei from the cell type of interest, from which chromatin is extracted and used to gain a cell type–specific view of TF occu-pancy, RNA Pol II distribution, cofactor recruitment, chromatin signatures or any chromatin-binding protein.

There are two important features of the BiTS method: (i) the embryos are covalently cross-linked before dissociation. This is crucial as it prevents any abnormal transcriptional response due to embryo/tissue dissociation and preserves DNA-protein interactions in their native context. Obtaining intact single cells from covalently cross-linked embryos or tissues is almost impossible, which has prohibited reliable FACS of cells from fixed tissue. However, single intact cross-linked nuclei can be readily dissociated from any fixed tissue or organism by mechanical disruption, centrifugation and filtration. (ii) FACS of fixed, labeled nuclei can obtain very high purities, typically in the range of 97% from a single sort, or > 99% from a double sort; this is a degree of purity that is difficult to obtain using biochemical methods. The sorting uses a standard FACS instrument and the conditions can be readily optimized for sorting nuclei from cells of different abundances. For example, for rare cell populations, a first ‘high-yield and low-purity’ fast sort to enrich the cell population followed by a second high purity sort will obtain the best recovery and accuracy.

Access to a cell sorter is the only real ‘limitation’ of the BiTS method that we are aware of, especially as sorting for a full day is desirable in order to include the setup of the FACS machine (~0.5–1 h) and the acquisition of a sufficient number of cell type–specific nuclei for several ChIP-seq experiments. For example, ~8 h of sorting yields ~40 million Drosophila mesodermal nuclei (at stages 10–11), which is sufficient for multiple ChIP experiments, as described below.

We have recently applied BiTS–ChIP-seq to study the relationship of histone modifications, TF and Pol II occupancy on develop-mental enhancers to enhancer activity during D. melanogaster mesoderm development25. These experiments show that BiTS-ChIP combined with next-generation sequencing generates cell type–specific data at very high sensitivity, specificity and resolu-tion. Notably, an analysis of TF occupancy showed that BiTS-ChIP provides reliable unskewed TF binding data that is comparable to standard ChIP-chip or ChIP-seq methods. Given the general properties of nuclei and FACS, BiTS should be widely applicable to organisms and tissues.

OverviewThe BiTS-ChIP method is designed to enable a cell type–specific analysis of transcriptional regulation (including TF and chromatin-binding protein occupancy as well as chromatin modifications) from heterogeneous biological samples in their native in vivo context. As outlined in Figure 1, the method involves four main modules: (i) expressing a nuclear tagged protein exclusively in the cell type of interest, (ii) obtaining thoroughly dissociated nuclei from covalently cross-linked embryos, (iii) FACS of fluorescent nuclei and (iv) chromatin isolation, immunoprecipitation and library preparation for next-generation sequencing.

To obtain tissue-specific nuclei, embryos expressing a tissue- specific nuclear marker are collected, aged to the desired developmental stage and cross-linked with formaldehyde to essentially ‘freeze’ the chromatin and regulatory context in situ. The marker used can either be an endogenous protein, a fluores-cent protein or an otherwise tagged nuclear protein specifically expressed in a cell population in transgenic animals (such as

Preparebiological material

Aldehyde fixation

Nuclei extraction

Primary antibody(mouse-anti-SBP)

Secondary antibody(anti-mouse:Alexa488)

FACSnuclear sorting

Concentratesorted nuclei

Extract chromatin

Shear chromatin(to ~200–250 bp)

Immunoprecipitation

Library preparation

Sequencing

Figure 1 | Schematic representation of the BiTS–ChIP-seq protocol. Staged embryos are bulk collected and fixed according to standard protocols, thereby stabilizing protein-DNA interactions. Fixed nuclei are extracted stained for cell type–specific expression of a marker protein. This step (gray text) can be avoided if a fluorescent protein such as GFPNLS is expressed in the cell type of interest. Fluorescent nuclei are then sorted on a regular FACS instrument to a purity of > 95%, and then the chromatin is extracted and sheared. The shearing of the chromatin allows for the subsequent immunoprecipitation of DNA associated with the protein (epitope) of interest. After purification of the immunoprecipitated DNA, the fragments can be used for library generation and subsequent deep sequencing or array hybridization.

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Drosophila embryos). Mechanical disruption of fixed embryos is used to dissociate and isolate nuclei; this is followed by several rounds of filtration and centrifugation to remove debris that might interfere with sorting. The ease of nuclei extraction from fixed, complex tissues is a key advantage of nuclear sort-ing compared with regular cell sorting. The accuracy and speed of FACS is used to identify and isolate the nuclei of interest to obtain a very pure population (typically >97%) of cell type–specific nuclei. The isolated nuclei are used for chromatin extraction, shearing and immunoprecipitation for applications ranging from an analysis of histone modifications, chromatin- binding proteins, TFs, DNA replication factors or general chromatin remodelers.

Experimental designCell type–specific labeling of nuclei. BiTS relies on the sorting of fluorescently labeled, fixed nuclei from the cell type or tissue of interest. This can be achieved using two methods:

A transgenic reporter expressed in the nuclei of the cell type of interest. Here we use a transgenic Drosophila line containing an epitope-tagged histone H2B driven by a mesoderm-specific enhancer/minimal promoter to fluorescently sort the nuclei of interest (Figs. 1 and 2). This approach requires staining of the dissociated nuclei prior to sorting. However, a nuclear-tagged fluorescent protein (for example, GFPNLS) should work equally well and this strategy would benefit from the vast com-pendium of transgenic animals already available in Drosophila, Caenorhabditis elegans, zebrafish and mice from enhancer- reporter studies. In Drosophila, researchers can also avail of the flexible Gal4-UAS system to direct specific expression that can even be temporally controlled.A highly specific antibody to an endogenous nuclear protein. If a very specific antibody is available for an endogenous nuclear protein that is exclusively expressed in the cell type of interest, transgenesis may not even be required, as was recently shown by nuclear sorting coupled to ChIP-seq of unfixed human tissue26.

Sorting strategies based on a tagged nuclear reporter expressed as a transgene will be most likely to give the highest degree of purity, given the specificity of enhancer-reporter expression, the variety of in vivo characterized enhancers available in most model sys-tems and the high quality of many tag-specific antibodies that can be used to fluorescently stain desired nuclei after fixation. When the nuclear protein used for staining is interacting with chroma-tin, preclearing of the antibody used for staining (Step 36 in the PROCEDURE) could in theory result in the depletion of certain chromatin regions. Although we did not observe this effect using

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tagged H2B25, we recommend using fluorescent proteins or a stain-able protein that does not interact with chromatin in the tissue/cell type of interest.

Strategies for expressing reporter genes. Expressing a reporter gene in a cell type of interest can be achieved by two strategies. First, if regulatory elements that control expression in the relevant cell type are known, these can be combined with a minimal pro-moter. The minimal promoter used should have no autonomous background activity and should only allow expression when it is combined with an enhancer of choice. For example, in Drosophila, we have made use of an eve minimal promoter, which shows no detectable basal activity and reliably drives gene expression when combined with tissue/cell-specific enhancer elements25. Alternatively, in some model organisms, versatile enhancer trap systems such as the Gal4-UAS system27 could be used. Second, in cases in which no known enhancer elements are available, it may be possible to use the endogenous locus of a gene that is expressed

a

100 µm

b

c

100 µmAnti-SBP

Presorting

Postsorting

Anti-SBP

Overshearing

Anti-SBP

d

Anti-SBP DAPI

Anti-SBP DAPI

Anti-SBP DAPI

Anti-SBP DAPI

Figure 2 | Mesoderm-specific nuclei are stained, extracted and sorted to purity. (a) Left, stages 9 and 10 embryos expressing a tagged nuclear protein in the mesoderm (twiPEMK::SBP-H2B). Chromatin is stained in DAPI (purple) and transgene expression is visualized using an anti-SBP antibody (green). Right, higher-magnification picture of the boxed region in the left image showing the posterior part of the embryo; note the nuclear localization of the marker protein (green). (b) Left, nuclei from twiPEMK::SBP-H2B–expressing embryos are extracted and stained for transgene expression (green, anti-SBP) and chromatin (right: purple, DAPI). At this stage, before sorting, only ~20% of all nuclei are mesodermal. (c) After sorting, >95% of all nuclei should be cell type (mesoderm) specific. (d) Overshearing of the nuclei from excessive douncing and/or syringing should be avoided, as it prohibits the efficient sorting of nuclei using FACS.

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in the tissue of interest by amending the coding region by homo-logous recombination with, for example, GFPNLS.

Collecting Drosophila embryos. Once transgenic lines or an appropriate marker have been established and the specificity of the expression exclusively in the cell type of interest is confirmed, biological material can be collected, covalently cross-linked and stored at − 80 °C for subsequent FACS. For Drosophila embryos, which are the focus of the remainder of this protocol, homozygous transgenic lines should be established if transgenes are being used, and the line expanded for collection. We routinely collect embryos from ten large cylindrical population cages ( 29 cm × 48 cm), each seeded with ~40 g of newly eclosed flies. After performing at least three 1-h pre-lays, 2-h embryo collections (using 15-cm molasses plates streaked with yeast paste) typically yield 3–5 g dry weight of tightly staged embryos, which are aged as desired and formalde-hyde cross-linked according to standard protocols. Information on how we collect and stage Drosophila embryos has been described in detail previously28. Fixed embryos can be stored at − 80 °C or used immediately.

Isolation of nuclei. Obtaining a clean nuclear preparation from frozen or fresh covalently cross-linked embryos is pivotal for the success of the BiTS-ChIP protocol and can usually be achieved by minimal optimization of existing nuclear extraction protocols. A good nuclear preparation should be devoid of contaminant cell clumps or other large matter and should not contain small subnuclear matter, such as broken nuclei and chromatin (Fig. 2b). High quality of the nuclear preparation is crucial to a successful sort, as sorting speed and fluidic stability depend on its purity.

Large cell clumps in the nuclear preparation, which could clog the nozzle of the sorter and cause instability of the flow, indicate insufficient mechanical disruption of the sample. The best solution to this problem is to increase the number of douncing steps and perhaps to use a higher-gauge needle for dissociation, followed by the removal of residual debris using a tight meshed filter (e.g., 20-µm Nitex membrane).

A less-frequent problem is the disruption of nuclear integrity and the appearance of subnuclear particulate matter and free- floating chromatin in the nuclear preparation (Fig. 2c). Breaking of nuclei is usually indicative of applying excessive force during the syringe steps in the protocol, or the use of very tight-gauge needles and can be easily overcome by reducing force or using lower gauge needles.

Chromatin fragmentation. Shearing success depends on the quality of fixation and the concentration of chromatin in the sample. Care should be taken not to vary the chromatin concentration too much between samples and to always start with a similar amount of sorted, cell type–specific nuclei. In all cases, the exact settings should be modified so as to achieve optimal shearing with minimal sonication; optimization can be performed using bulk, unsorted nuclei.

Chromatin size must be considered in light of downstream appli-cations; whereas approximately 200–250-bp shearing performs well for Solexa sequencing, other applications such as microarray ana-lysis may require larger chromatin fragments. In our case, we used 40 million sorted nuclei in 300 µl of immunoprecipitation buffer supplemented with fresh proteinase inhibitors. By using a Diagenode Bioruptor precooled to 4 °C, ~200–250 bp shearing

was achieved in 1.5-ml low binding tubes in the appropriate tube adapter with 18 high-energy cycles of 30 s ON/30 s OFF. If the Bioruptor is not actively cooled, ice-cold water should be exchanged every 5–6 sonication cycles, but the bath should be free of ice.

ChIP optimization. An essential step for performing ChIP, whether BiTS-ChIP or standard ChIP, is to optimize the IP conditions for each antibody individually. IP conditions depend on the quality of the antibody and the abundance and accessibility of the target protein epitopes. The optimal amount of chromatin, antibody and beads should be determined on unsorted material with two con-siderations in mind—enrichment and recovery.

Enrichment provides an indication of the specificity of the ChIP and can be monitored in real-time PCR analyses of expected enriched regions over suspected negative regions. The enrichment determined serves as a guide in finding which antibody, chromatin and bead amounts allow for the best enrichment.

Recovery provides an indication of the efficiency of the ChIP and is especially important for subsequent library generation (dis-cussed below). The total DNA yield of an IP depends largely on two parameters. First, proteins (or modifications) might cover a small subset of the genome (e.g., TFs, H3K4me3), or may be dis-tributed over a large portion of the total genome (e.g., H3K4me1, H3K27me3), which naturally correlates with the total DNA yield. Second, antibodies can differ substantially in their quality. Other important factors affecting the recovery are the washing conditions and the amounts of beads used. Washes that use high-salt buffers typically result in increased enrichment and reduced recovery. If the recovery is too low for library generation, washing times can be shortened and high-salt washes substituted for lower-salt washes, although this runs the risk of decreasing the specificity and thereby increasing the background signal.

These considerations affect the amount of chromatin needed for a successful ChIP-seq experiment and need to be adjusted for optimization. Table 1 provides information on the IP conditions we used for different antibodies on BiTS-isolated chromatin25, which could serve as a starting point for IP optimization in other systems.

Library generation. In general, library generation is highly dependent on the amount of DNA supplied. The library protocol described here requires a minimum of 2 ng of DNA, but it works best with 5–10 ng of material. In case the amount of immuno-precipitated DNA material is very low, other library protocols can be tested which have been optimized for as little as 30 pg of chromatin29,30.

An important concern when starting with low amounts of DNA (<2 ng) is the introduction of artifacts by, for example, excessive PCR amplification of the material. It is generally advisable to use the fewest PCR cycles possible, and we routinely use 15–18 cycles. The resulting library eluate should be in excess of 2 ng µl − 1, and we routinely have concentrations of 20–50 ng µl − 1. We find that we obtain much better results by performing a second gel isolation after PCR amplification. Although this does introduce consider-able loss of material, the advantage is that it allows for very reliable exclusion of unincorporated primers, primer dimers and short artifactual amplicons. The second gel isolation can be done with a blade to allow for maximal recovery of the PCR-amplified material, which is readily visible under blue light.

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How many nuclei do I need for an experiment? The amount of sorted nuclei needed for downstream analysis depends largely on the application, but is generally considerably less than what would be needed when probing entire embryos for epitopes that are only present in a subset of cells. For example, when examining genome-wide occupancy of a mesodermal TF (Mef2) in develop-ing Drosophila embryos, we used only 15 µg of chromatin from sorted nuclei (where the TF is expressed) compared to 40 µg of chromatin from the whole embryo25. The most important con-sideration with respect to how much chromatin is needed for a given experiment is the efficiency of the ChIP itself. Although ChIP using antibodies directed against histones or histone modifications may recover up to 20%, ChIP of TFs or other DNA-binding pro-teins is typically less efficient, with recovery of as little as < 0.1% of input material for some factors.

Three to four micrograms of chromatin are normally obtained from about 4–6 million sorted nuclei in D. melanogaster. We gener-ally opt to sort nuclei from embryos in large batches, starting with ~1–1.5 g dry weight of formaldehyde cross-linked embryos, thereby obtaining enough chromatin for several experiments. Sorting of embryonic mesodermal nuclei, which constitutes ~20% of the total input material, over a period of ~8 h typically results in ~40–50 million sorted nuclei, generating ~30 µg of cell type–specific chro-matin. Using our optimized ChIP protocol, we use between 3 and 5 µg of chromatin for ChIP experiments for widespread, abundant

chromatin modifications (e.g., H3K4me1), and 10–15 µg of chro-matin for DNA-binding proteins such as TFs or Pol II (Table 1).

Adapting the protocol to other model organisms. One of the big-gest advantages of the BiTS–ChIP-seq protocol is its simplicity and general applicability. To perform BiTS-ChIP in another system, a cell type–specific way to mark the nuclei of the cell type of inter-est is required. BiTS can take advantage of the vast array of exist-ing transgenic lines expressing a fluorescent nuclear protein or an epitope-tagged protein in the cell types of interest. Alternatively, a specific antibody directed against an endogenous nuclear protein can be used to fluorescently label specific cell types (e.g., NeuN, which is specific for neurons)31.

The nuclear extraction protocol from fixed embryos is the step that is most likely to require some species-specific optimization. A prerequisite for this is to first optimize the fixation conditions for standard ChIP in that species. The nuclear extraction step from fixed embryos should be readily achieved by modifying existing nuclear extraction protocols (e.g., adjusting douncing and syring-ing procedures and including filtration steps for large particulate matter as described in this protocol).

Once a clean nuclear preparation is obtained, sorting of fixed nuclei can be performed as described here, as the general properties of nuclei and parameters of FACS are expected to be the same for most model organisms.

taBle 1 | IP conditions.

antibodychromatin

(mg)pas slurry (50%; ml) Washes

Ip yield (ng)

H3 (Abcam, ab1791), 2.5 µl 3 10 Regular: 1× RIPA, 4× RIPA500, 1× LiCl, 2× TE buffer (10 min each)

~120

H3K4me1 (Abcam, ab8895), 3 µg 5 10 Regular: 1× RIPA, 4× RIPA500, 1× LiCl, 2× TE buffer (10 min each)

~50

H3K27me3 (Active Motif, 39155), 3 µg 5 10 Regular: 1× RIPA, 4× RIPA500, 1× LiCl, 2× TE buffer (10 min each)

~40

H3K36me3 (Abcam, ab9050), 3 µg 5 10 Regular: 1× RIPA, 4× RIPA500, 1× LiCl, 2× TE buffer (10 min each)

~30

H3K79me3 (Abcam, ab2621), 3 µg 5 10 TE quick: 1× RIPA, 4× RIPA500, 1× LiCl (10 min. each), 2× TE buffer (5 min each)

~10

H3K27ac (Abcam, ab4729), 3 µg 10 10 TE quick: 1× RIPA, 4× RIPA500, 1× LiCl (10 min each), 2× TE buffer (5 min each)

~20

H3K4me3 (Abcam, ab8580), 2 µg 10 10 TE quick: 1× RIPA, 4× RIPA500, 1× LiCl (10 min each), 2× TE buffer (5 min each)

~25

Rpb3 (John Lis Lab), 4 µl (serum) 15 10 Soft: 5× RIPA (6 min each), 1× LiCl (1 min), 2× TE buffer (1 min)

~15

Mef2 (E. Furlong Lab), 4 µl (serum) 15 10 Soft: 5× RIPA (6 min each), 1× LiCl (1 min), 2× TE buffer (1 min)

~10

Summary of the IP conditions as used in Bonn et al.25. Described are the antibodies used, the amount of input chromatin (Chromatin in µg), the amount of Protein A–Sepharose (50% slurry in µl), the washing conditions, and the final yield of immunoprecipitated chromatin (IP yield in ng).

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MaterIalsREAGENTS

HB buffer (Tris-HCl, sucrose, NaCl, KCl, EDTA, EGTA; see REAGENT SETUP)Complete proteinase inhibitors (Roche, cat. no. 11873580001)PBT buffer (Triton X-100, PBS; see REAGENT SETUP)PBTB buffer (PBT, BSA; see REAGENT SETUP)Flow-Check fluorospheres (Beckman Coulter, cat. no. 6605359)BD FACSFlow sheath (Becton Dickinson GmbH, cat. no. 342003)DAPI (Cell Signalling Technology, cat. no. 4083) ! cautIon DAPI is toxic and a mutagen. Gloves should be always worn while working with DAPI.RIPA buffer (NaCl, EDTA,Triton X-100, SDS, sodium deoxycholate, Tris-HCl; see REAGENT SETUP)TE buffer (Tris-HCl, EDTA; see REAGENT SETUP)Protein G–Sepharose CL4B (Sigma, cat. no. P3296)Protein A–Sepharose CL4B (Sigma, cat. no. P9424)Bovine serum albumin (BSA; Sigma, cat. no. A7906)RIPA500 buffer (NaCl, EDTA,Triton X-100, SDS, sodium deoxycholate, Tris-HCl; see REAGENT SETUP)LiCl buffer (LiCl, EDTA, IGEPAL CA-630, sodium deoxycholate,Tris-HCl; see REAGENT SETUP)RNase A (Qiagen, cat. no. 1006693)Proteinase K (Roche, cat. no. 745723)Phenol/chloroform/isoamylalcohol (Ambion, cat. no. 9732) ! cautIon Phenol is highly toxic and should only be used in the fume hood. Wear gloves and a lab coat when handling phenol.Chloroform (Sigma, cat. no. C2432) ! cautIon Chloroform is toxic and should only be used in the fume hood. Wear gloves and a lab coat when handling chloroform.Glycogen (Roche, cat. no. 901393)Quant-iT dsDNA assay kit (Life Technologies, cat. no. Q33120)NEBNext ChIP-seq sample prep master mix set (NEB, cat. no. E6240S/L)QIAquick MinElute PCR purification kit (Qiagen, cat. no. 28004)Paired-end Adapters (Illumina, cat. no. 1001782)GelGreen nucleic acid gel stain (Biotium, cat. no. 41005) ! cautIon GelGreen is toxic and gloves should be always worn while working with it.QIAquick gel extraction kit (Qiagen, cat. no. 28704)Phusion high-fidelity PCR kit (NEB-Finnzymes, cat. no. F553L)PCR Primer PE 1.0 (Illumina, cat. no. 1001783)PCR Primer PE 2.0 (Illumina, cat. no. 1001784)MinElute gel extraction kit (Qiagen, cat. no. 28604)Mouse anti-SBP antibody (Santa Cruz Biotechnology, cat. no. 101595)Anti-mouse Alexa Fluor 488 (Alexa488) antibodyBD FACSRinse solution (BD Biosciences, cat. no. 340346) or BD FACS clean solution (BD Biosciences, cat. no. 340345)

EQUIPMENTDounce homogenizer (15 ml; Wheaton Scientific)Miracloth (Calbiochem, cat. no. 475855)Needles (BD (20G), cat. no. 301300; (22G), cat. no. 300900)Nitex membrane (20 µm; SEFAR, cat. no. 03-20/14)Neubauer chamber (Assistent, cat. no. 442/12)Cell strainer (70 µm; BD Falcon, cat. no. 352350)Polypropylene round-bottom tubes (BD Falcon, cat. no. 352063)Cell sorterLow-binding reagent tubes (1.5 ml; Biozym, cat. no. 710176)Bioruptor water bath sonicator with 1.5-ml tube holder (Diagenode)Phase-lock heavy gel tubes (2 ml; Eppendorf, cat. no. 0032-005-152)Qubit fluorometer (Life Technologies, cat. no. Q32866)SafeXtractor (100 tips; 5PRIME, cat. no. 2600010)2100 Bioanalyzer (Agilent Technologies)Agilent DNA 1000 kit (5067-1504)MoFlo cell sorter (Beckman Coulter)Illumina Solexa system (or outside provider)Serological pipettesFilter, 22-µm pore (Millipore, cat. no. SLGV033RS)

REAGENT SETUPCollection and formaldehyde cross-linking of biological material For Drosophila embryos, collect, stage and cross-link material in 1.8% (wt/vol) formaldehyde for 15 min, according to Sandmann et al.28; per sort, we use

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dry-weight embryos of 1–1.5 g, but the amount of cell type–specific nuclei depends on both cell density and the relative abundance of the sorted cell type within the embryo. crItIcal The collection of biological material should be performed in advance. Formaldehyde cross-linked Drosophila embryos can be stored at − 80 °C for at least 1 year.HB buffer To prepare HB buffer, mix 15 mM Tris-HCl (pH 7.4), 0.34 M sucrose, 15 mM NaCl, 60 mM KCl, 0.2 mM EDTA and 0.2 mM EGTA. This solution can be stored at 4 °C for 1 year when filter-sterilized. crItIcal Proteinase inhibitors (Roche Complete) should be added shortly before use (1 tablet per 50 ml buffer).PBT buffer This buffer consists of 0.1% (vol/vol) Triton X-100 in PBS. The solution can be stored at room temperature (25 °C) or at 4 °C for 1 year.PBTB buffer Add 5% (wt/vol) BSA to PBT buffer. crItIcal This solution should be freshly prepared on the day of use and kept at 4 °C. Proteinase inhibitors should be added shortly before use (Roche Complete, 1 tablet per 50 ml) and the solution must be filtered through a 0.22-µm-pore filter, as undissolved albumin particulates may affect sorting.RIPA buffer To prepare RIPA buffer, combine 140 mM NaCl, 1 mM EDTA, 1% (vol/vol) Triton X-100, 0.1% (wt/vol) SDS, 0.1% (wt/vol) sodium deoxycholate and 10 mM Tris-HCl (pH 8.0). This solution can be stored at 4 °C for 1 year.TE buffer To prepare TE buffer, mix 1 mM EDTA and 10 mM Tris-HCl (pH 8.0). This solution can be stored at room temperature or 4 °C for 1 year.Protein G–Sepharose CL4B and/or protein A–Sepharose CL4B Bead suspension (slurry; 50% (vol/vol)) should be prepared in advance. In brief, resuspend beads in an equal volume of PBS (80%)/ethanol (20%) (vol/vol) and transfer it into a 15-ml conical tube. Centrifuge at 1,000g for 2 min at room temperature to pellet the beads. Aspirate the supernatant carefully, add PBS (80%)/ethanol (20%) to obtain a 50% (vol/vol) beads slurry. Make 1-ml aliquots, which can be stored at 4 °C for 1 year.RIPA500 buffer RIPA500 buffer is prepared by combining 500 mM NaCl, 1 mM EDTA, 1% (vol/vol) Triton X-100, 0.1% (wt/vol) SDS, 0.1% (wt/vol) sodium deoxycholate and 10 mM Tris-HCl (pH 8.0). This solution can be stored at 4 °C for 1 year.LiCl buffer Combine 250 mM LiCl, 1 mM EDTA, 0.5% (vol/vol) IGEPAL CA-630, 0.5% (wt/vol) sodium deoxycholate and 10 mM Tris-HCl (pH 8.0). This solution can be stored at 4 °C for 1 year.Proteinase K solution Resuspend proteinase K in 20 mM Tris-HCl (pH 7.4), 1 mM CaCl

2 and 50% (vol/vol) glycerol. Proteinase K solution can be stored

at − 80 °C for 1 year.EQUIPMENT SETUPSetup for chromatin fragmentation The setup time for chromatin fragmen-tation is dependent on the device used (e.g., Bioruptor or Covaris AFA). If a Bioruptor with a cooling device is used for chromatin fragmentation, the device should be cooled down at least 30 min before use to reach the proper working temperature (4 °C). If the Bioruptor is not equipped with a cooling device, sonication can be performed by supplying fresh ice-cold water every five sonication cycles. If a Covaris AFA is used for chromatin fragmen-tation, the instrument needs to be turned on 30–60 min before use to degas the water, which is crucial for reliable fragmentation.MoFlo cell sorter configuration for nuclei sorting ● tIMInG 0.5–1 h The cell sorter should be equipped with a laser generating 200 mW of 488-nm TEM

00 mode; an example is a Coherent Innova 90C argon-ion laser. Both

the sample station and the sample collection module should be refrigerated at 4 °C during the entire duration of the procedure. For maximum droplet generation and sorting speed, a 70-µm nozzle tip (around 95 kHz droplet generation at 60 psi of sheath pressure) is recommended when sorting nuclei. The sheath fluid (BD FACSFlow) is filtered in-line through a Fluorodyne II 0.2-µm filter (Pall)

Sorting tissue-specific nuclei from embryo lysates is time consuming, and we recommend filling and pressurizing the sheath tank the day before the experiment to allow time for air/fluid equilibration in the system. This cuts the instrument setup time considerably. We also recommend cleaning and inspecting the ceramic nozzle the day before to rule out sediments or particles that might inhibit fluidic stability. Orthogonality of the laser beam and the fluidic jet should be confirmed in order to maximize instrument

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performance (for details of our in-house laser beam horizontal alignment tool for the MoFlo, please check the tools section at http://www.fccf.embl.de/fccfweb/). Flow-Check fluorospheres should be used in fine alignment of laser, stream, optical filters, mirrors and photomultiplier tubes (PMTs), as well as in calculations of the instrument drop delay. Original MoFlo’s L-configuration could be used as the optical layout of the primary laser (Fig. 3). Band-pass (BP) filters (530/40 and 670/40 nm) should be used in the collection of green and red fluorescence in the 488-nm laser path. Optionally, a StopLine Holographic Notch 488 filter (OptoSigma) can be placed in the optical layout to prevent unwanted laser noise from reaching the fluorescence detectors.

During the instrument setup, data acquisition should be triggered on forward scatter (FSC) with no threshold applied. Alignment setup with beads should also guarantee maximum FSC light arrival at the photodiode detector. By manually setting the laser stop bar in front of the FSC collection lenses to a minimum width and preventing laser spillover and droplet generation scatter noise from reaching the detector, we can ensure that the small scatter-ing properties of the nuclei are detectable (see TROUBLESHOOTING). The FSC collection lens assembly’s position needs to be finely adjusted for

maximum light collection and low background. Place neutral-density filters in the path to photodiode and PMTs if attenuation of the Flow-Check fluorosphere signal is required. These neutral density filters should be removed before acquisition and sorting of nuclei. Calculate the instrument drop delay (see instrument manufacturer’s manual).

FL2

670/40 BP

555 DCLP

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488/

10 B

P

488/

10 B

P

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SSC

FL1Alexa488

Notch 488

200 mW488-nm Laser line

530/

40 B

P

Figure 3 | Cell sorter configuration used in the acquisition and sorting of mesodermal D. melanogaster nuclei. Sample acquisition on the MoFlo was triggered on photodiode-collected FSC signals (488/10-nm BP). Alexa488 fluorescence from positive nuclei exposed to 200 mW of a 488-nm laser was collected by the instrument’s photomultipliers after filtering with 530/40 (green)-nm and 670/40 (red)-nm band-pass filters. A StopLine Holographic Notch 488 filter (OptoSigma) was placed in the path to the fluorescence detectors in order to block unwanted laser noise.

proceDure crItIcal Unless indicated otherwise, centrifugation and incubation steps are always performed at 4 °C and on ice.

extraction of nuclei ● tIMInG ~1 h1| Transfer 1–1.5 g of frozen embryos, collected and fixed as published28, into a 15-ml dounce homogenizer. Add 10 ml of chilled HB buffer per gram embryos and incubate for 5 min on ice.

2| Dissociate the embryos by pipetting up and down with a 10-ml serological pipette.

3| Dounce 20 times with a loose pestle and 10 times with a tight pestle. crItIcal step This step frees the nuclei from the rest of the material, and thus care should be taken to get a homogenous solution. If the embryos are not dounced sufficiently, the nuclear yield will be decreased.

4| Filter the lysate through two layers of Miracloth into a 15-ml conical tube. The Miracloth layers should be rotated by 90° to each other’s grain and placed into a plastic funnel. Squeeze the cloth gently to drain the lysate.

5| Spin at 3,500g for 5 min to pellet the nuclei, and then carefully pour off the murky supernatant. crItIcal step Care should be taken not to solubilize the hard, yellow yolk pellet, which may form at the bottom of the tube.

6| Wash the nuclei in 10 ml of HB buffer, pipette up and down with a 10-ml serological pipette to dissociate the nuclei, and transfer them to a new 15-ml conical tube. Pellet the nuclei at 3,500g for 5 min.

7| Resuspend the nuclei in 3 ml of PBTB buffer and transfer them to a 15-ml conical tube.

8| Dissociate the nuclei by passing them ten times through a 20-G needle and then ten times through a 22-G needle using a 5-ml syringe. crItIcal step Do not apply too much pressure when syringing to avoid shearing and excessive foaming.

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9| Wash a square of 20-µm Sefar Nitex membrane with deionized H2O to remove particulates. The square should be cut large enough that it covers the opening of a small plastic funnel.

10| Place the funnel into a fresh 15-ml conical tube, and then place the Nitex membrane on top and hold it there with light tension. Pipette the nuclei from Step 8 through the Nitex membrane by placing a 5-ml serological pipette directly onto the membrane; expel the solution of nuclei through the membrane and into the 15-ml conical tube.

11| Estimate the total number of nuclei and verify their effective dissociation by microscopy. A small aliquot should be stained, for example with DAPI, and examined in a Neubauer chamber.! cautIon DAPI is toxic and a mutagen. Gloves should be always worn while working with DAPI. crItIcal step A 10-µl quality control aliquot should be serially diluted (for example, 1:10 and 1:30, depending on the initial concentration of nuclei in the sample). Nuclei should not appear to cluster in clumps and should be intact and well defined; DAPI-positive subnuclear debris is indicative of excessively sheared nuclei and such samples should be discarded (compare Fig. 2b with Fig. 2d).? trouBlesHootInG

cell type–specific nuclear staining (optional) ● tIMInG ~2.5 h crItIcal Staining is only needed if no fluorescent nuclear marker such as GFP has been used to label the nuclei in the cell type of interest. If a fluorescent marker has been used, Steps 12–15 can be omitted, but staining may be used to enhance a weak fluorescent signal.

12| Stain nuclei with appropriate primary antibodies. We stained a histone protein fused to a SBP tag, specifically expressed in the cell type of interest, with a mouse anti-SBP antibody at 1:100 for 1 h, nutating at 4 °C. crItIcal step The appropriate dilution factor for the primary antibody needs to be optimized for each antibody.

13| Add 6 ml of PBTB, spin for 2 min at 1,000g and discard the supernatant. This step removes most of the free primary antibody.

14| Add a fluorescent secondary antibody. We used anti-mouse Alexa488 antibody at 1:100 in 3 ml of PBTB and incubated for 1 h while nutating at 4 °C. Completely resuspend nuclei by pipetting up and down. crItIcal step Solutions containing fluorescent antibodies should be protected from light to avoid bleaching of the fluorophore. The appropriate dilution factor for the secondary antibody needs to be optimized for each antibody.

15| (Optional) Estimate the proportion of desired nuclei in the input sample (Fig. 2). Counterstain a small aliquot with DAPI, and then serially dilute and examine it by epifluorescence. Determine the ratio of fluorescent + DAPI nuclei to nonfluorescent + DAPI nuclei.! cautIon DAPI is toxic and a mutagen. Gloves should be always worn while working with DAPI.

sorting of labeled cell type–specific nuclei ● tIMInG variable16| Invert the nuclei from Step 10 (or Step 15 if optional staining has been performed) in PBTB repeatedly to mix. Transfer 300 µl of the suspension into 1.5-ml microcentrifuge tubes containing 1 ml of PBTB buffer.

17| Pellet the nuclei in each tube by centrifugation at 1,000g for 3 min. Carefully discard the supernatant, resuspend the nuclei in 1 ml of PBTB, and transfer the nuclear suspension to a 5-ml polypropylene round-bottom tube. Bring the suspension to 3 ml with PBTB. crItIcal step Nuclei FACS conditions described in Steps 16–26 are for fixed nuclei, stained with Alexa488 fluorescent antibodies. If the nuclei sample was stained, the supernatant after centrifugation contains free unbound Alexa488- conjugated antibodies, which, if not removed completely will increase the background signal and lower sorting efficiency.

18| Just before the sort, resuspend the nuclei by passing the solution approximately seven times through a 22-G needle with a 3-ml syringe.

19| Filter the nuclei suspension through a 70-µm cell strainer into a 5-ml polypropylene round-bottom tube. crItIcal step Steps 18 and 19 disrupt nuclear aggregates that form by sedimentation, allowing for better sort yield and preventing instrument clogging.

20| Run the nuclei sample from Step 19 in a FACS machine and plot 530/40 BP fluorescence (log) against FSC (area).

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21| Adjust both FSC gain and 530/40 BP PMT voltage in order to bring 530/40-negative and 530/40-positive nuclei on scale. A contaminant population of dimmer green autofluorescence and smaller FSC than nuclei should appear in the left corner of the plot.

22| Gradually increase the FSC threshold until the instrument detects as few FSC subnuclear events as possible, while keeping the nuclei populations well over threshold.

23| Define the sorting gate as a combination of regions drawn around Alexa488 (530/40)-positive nuclei and singlet events (for instance, SSC-log versus Alexa488-log, Alexa488-log versus 670/40-log and FSC-area versus pulse width; Figs. 3 and 4).? trouBlesHootInG

24| Increase the event rate to 3 × 104 s − 1 or higher. Sort Alexa488 + nuclei under Purify 1.0 mode, 4 × 106 nuclei at a time into cooled 5-ml tubes (or similar) containing 500 µl of PBTB buffer. crItIcal step Check that the instrument ′sort abort rate′ is below 10% of the frequency of sorts.? trouBlesHootInG

25| Aliquot 10 µl (~10,000 nuclei) for purity assessment at the beginning of the sorting day and after each sorting round of 4 × 106 nuclei. crItIcal step Splitting the sorted nuclei into fractions is recommended in long procedures as a way to limit contaminations due to transient instability in the fluidics, unexpected nozzle clogging and drop delay shifts.

26| Evaluate the purity of the fractions after each round of 4 × 106 nuclei by DAPI counterstaining the aliquot taken in Step 25 and inspecting by epifluorescence microscopy. The average purity of the sorted fractions (DAPI + Alexa488 + ) should be at least 95% (Fig. 2).! cautIon DAPI is toxic and a mutagen. Gloves should be always worn while working with DAPI. crItIcal step Systematic assessments of the purity of the sort fractions will be used to monitor the sort quality along the day. Low-purity fractions should be discarded.? trouBlesHootInG

lysis of the nuclei and shearing of the chromatin ● tIMInG ~1 h27| Combine all fractions of at least 95% purity in a 50-ml conical tube. Make note of the number of sorted nuclei that will be processed further, as well as of the overall purity.

1,000

0

2,000

3,000

4,000

1010 102 103 104

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C-a

rea

Alexa488-log (530/40 BP)

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101

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9.79

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1010 102 103 104S

SC

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1,0000 2,000 3,000 4,000

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1,0000

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Pulse width

c

101

1010

0

102

102

103

103

104

104

FL2

-log

(670

/40

BP

)


e

Figure 4 | Representative flow cytometric data, regions and sort gate. (a) FSC collection lens and the laser stop-bar were finely adjusted during the initial acquisition of the nuclei sample while simultaneously inspecting Alexa488 versus FSC-area data. A FSC threshold was applied in order to cut down subnuclear contaminants (bottom left population). (b) A region drawn around the Alexa488 nuclei population in 530/40 BP versus SSC-log plots (b–d) was used to identify intact single nuclei in pulse width (c) and scatter plots (d). (e) A green (530/40 BP) versus red (670/40 BP) fluorescence plot was used to define Alexa488 nuclei from autofluorescent events. These regions were combined as the sort decision gate.

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28| Pellet the nuclei by centrifugation at 3,500g, 4 °C for 10 min and remove the supernatant. crItIcal step It is important to remove as much liquid as possible, as the nuclei will be resuspended in a relatively small volume.

29| Resuspend the nuclei in 300 µl of RIPA buffer (supplemented with protease inhibitors) by pipetting with a 200-µl pipette tip about five times and transfer the suspension to a 1.5-ml low-binding tube.

30| Incubate the suspension on ice for 10 min.

31| Sonicate the sample in order to shear the chromatin to an average fragment size of ~200–250 bp (see INTRODUCTION and Fig. 5a).! cautIon Always wear ear protection when operating the sonicator.

32| Centrifuge the sonicated chromatin for 2 min at 18,000g and transfer the supernatant into a new 1.5-ml low-binding tube. Aliquot ~30 µl for quality assessment (QA sample). pause poInt The chromatin can be stored at − 80 °C for at least 1 year after flash-freezing in liquid N2.

33| To determine the DNA yield and fragment size distribution, take the QA sample and treat it as described in Steps 45–54. Determine the concentration of DNA spectrophotometrically and inspect the size distribution by gel electrophoresis using 1.5–2% (wt/vol) agarose gels. The Bioruptor settings described generate an average fragment length of ~200 bp (Fig. 5a), which is optimal for subsequent ChIP-seq analysis. crItIcal step If the fragmentation conditions are reliably set, the QA sample can be retained and processed with the IP samples (Steps 34–44) at Step 45. If the fragmentation size of chromatin is variable, it is advised to first examine the size of the chromatin before proceeding with Step 34.? trouBlesHootInG

preabsorption and immunocomplex formation ● tIMInG 1 d crItIcal The preabsorption is needed if the sample has been antibody-stained in order to remove the antibody complexes generated by the staining procedure. If a fluorescent protein such as GFP was used for sorting, preabsorption can reduce nonspecific binding of chromatin to Protein G (or Protein A)–Sepharose, but it is not strictly necessary and Steps 34–36 can be omitted. The choice of bead material depends on the antibodies used (staining and IP), as antibodies raised in different species differ in their affinity to Protein G or Protein A. Here we describe the use of Protein G beads for preclearing, as the mouse-derived anti-SBP antibody we used shows stronger affinity for Protein G than for Protein A. For IP we use Protein A beads, as they have strong affinity for the rabbit-derived polyclonal antibodies used here (table 1). Note that this protocol is for 40 million sorted nuclei, which in D. melanogaster corresponds to ~25 µg of DNA. Steps 34–54 are similar to what has been described previously28.

34| Wash 25 µl of Protein G–Sepharose slurry twice with 1 ml RIPA. Centrifuge at 1,000g for 2 min and discard the supernatant. Resuspend the beads in 100 µl of RIPA buffer.

1.0

kb

Size selectionChromatin

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M MS M MS M MS M MS

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15 1,500 700500400300200100 bp

Lowermarker(lm)

Uppermarker(um)

Size: 283 bpConcentration: 41.6 ng µl–1

Molarity: 424.2 nmol l–1

Um

Lm

a

b

Figure 5 | Solexa sequencing library preparation from tissue-specific, sheared chromatin. (a) For Solexa sequencing, tissue-specific chromatin should be sheared to an average size of ~200 bp (QA sample, Steps 31 and 33 ‘chromatin’). For library generation, the adapter-ligated immunopurified DNA is run on an agarose gel, and then a gel slice is cut out for size selection (here: chromatin size of ~200 bp plus 40–50 bp to account for adapters; Step 68, ‘size selection’). The size-selected material is PCR amplified (Step 73, ‘library PCR’); this is followed by a second gel purification (Step 77, ‘purification’). This final purification excludes unicorporated primers, primer dimer products and PCR artifacts (asterisks). (b) The final product should be quality assessed on a Bioanalyzer before sequencing. The peak should show a narrow normal distribution, centered around the expected size of ~250–300 bp.

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35| Take the chromatin from Step 32 (~300 µl) and adjust the final volume to 900 µl with RIPA buffer. crItIcal step Depending on the abundance of the target epitope and the quality of the antibody, we use between 3 and 15 µg of DNA for a single IP (table 1). The material can thus be used for several IPs.

36| Add the bead suspension from Step 34 to the 900 µl of chromatin from Step 35. Incubate on a rotating wheel at 4 °C for 1 h and pellet the beads by centrifugation at 1,000g at 4 °C for 2 min. crItIcal step The Protein G–Sepharose is used for preabsorption, which is vital if the sample is stained, as the antibody used for staining could otherwise interfere with the IP reaction.

37| Transfer the supernatant to a new 1.5-ml low-binding tube and avoid any bead carryover. Retain 30 µl of the sample (input control sample) in a separate tube and store at 4 °C until Step 45. crItIcal step Always use low-binding tubes for steps involving low amounts of DNA to minimize loss from nonspecific binding of the chromatin to tube walls.

38| For IP, add a suitable amount of antibody or serum to a predetermined amount of precleared sample from Step 37 and incubate the mixture overnight on a rotating wheel. crItIcal step The serum or antibody used for the IP should be directed against the epitope of interest (protein or protein modification) and the choice is limited by the availability of ChIP-grade antibodies. In our case, we used antibodies against TFs (for example, Mef2), a subunit of a protein complex (for example, Rpb3), and histone modifications (for example, H3K4me3; see INTRODUCTION and table 1).

39| For each precipitation, wash 10 µl of 50% Protein A suspension once with 1 ml of RIPA buffer supplemented with 1 mg ml − 1 BSA. Pellet the beads by centrifugation at 1,000g for 2 min and incubate in 1 ml of RIPA buffer supplemented with 1 mg ml − 1 BSA on a rotating wheel or nutator at 4 °C overnight. crItIcal step This step reduces the unspecific binding of chromatin to the Protein A beads. The optimal amount of bead slurry used needs to be tested beforehand (see INTRODUCTION and table 1).

purification of immunocomplexes ● tIMInG 1 d40| Centrifuge the preblocked Protein A beads at 1,000g for 1 min, remove the supernatant and resuspend in 100 µl of RIPA buffer per reaction.

41| Purify immunocomplexes by adding 100 µl of preblocked PAS solution to the antigen-antibody complexes from Step 38 and incubate for 3 h on a rotating wheel.

42| To purify the complexes, pellet the beads by centrifugation at 1,000g for 1 min, dispose of the supernatant and rinse the beads once with 1 ml of RIPA buffer.

43| Pellet the complexes again by centrifugation at 1,000g for 2 min and remove the supernatant.

44| Wash the beads for 10 min on a rotating wheel in the following buffers: once in RIPA, four times with RIPA500, once with LiCl and twice with TE. After each wash, centrifuge as in Step 43 and discard the supernatant. crItIcal step The washes should be adjusted to the antibody-antigen affinity. In general, for IPs for chromatin modifications the general protocol can be used. For more transient interactions such as DNA-protein complexes (for example, IPs for Pol II), the washing parameters should be optimized. We often reduce the length of washes for LiCl and TE, if necessary. Also, the RIPA500 washes could be changed to standard RIPA washes (lower salt). The exact IP conditions should be optimized on unsorted material beforehand (see INTRODUCTION and table 1).

45| From this point on, include also the QA and input control samples (Steps 33 and 37, respectively); adjust the volume to 100 µg with TE buffer. Add RNase A to 50 µg ml−1 and incubate at 37 °C for 30 min.

46| Adjust the samples to 0.5% (wt/vol) SDS, 0.5 mg ml − 1 proteinase K and incubate them at 37 °C overnight.

cross-link reversal and Dna purification ● tIMInG 8 h47| Incubate the samples at 65 °C for 6 h to reverse the formaldehyde cross-links.

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48| Add 100 µl of TE to each sample. crItIcal step This step increases the total volume of the reaction and decreases the pipetting loss during the subsequent chloroform extraction.

49| Extract the DNA by combining the sample with 300 µl of phenol/chloroform/isoamylalcohol in a precentrifuged 2-ml Eppendorf Phase-lock tube, mix by inversion 5 times and centrifuge at > 15,000g for 5 min at room temperature. Add 300 µl of fresh chloroform, invert about five times to mix and centrifuge the tube at 15,000g for 5 min at room temperature. Transfer the aqueous phase sample (~200 µl of the upper phase) to a fresh low-binding reaction tube.! cautIon Phenol and chloroform are both toxic and should only be used in a fume hood. Wear gloves and a lab coat when handling phenol and/or chloroform.

50| Add 0.25 mg ml − 1 glycogen, 25 µl of 3 M sodium acetate (pH 5.2) and 550 µl of ethanol to the samples; vortex thoroughly and incubate the samples at − 80 °C for 30 min.

51| Centrifuge the sample for 30 min at 15,000g at 4 °C to precipitate the DNA, wash the pellet once with 500 µl of 70% (vol/vol) ethanol and centrifuge again at 15,000g at 4 °C for 5 min.

52| Remove the supernatant carefully, air-dry the pellet and resuspend it in 30 µl of TE. crItIcal step Make sure not to disturb the whitish pellet of glycogen and DNA. Dry the pellet until no ethanol is visible, as residual ethanol can interfere with subsequent library preparation for Solexa sequencing (Steps 55–81).

53| Measure the DNA concentration of the immunoprecipitated material using a Qubit fluorometer and dsDNA high sensitivity assay reagents. crItIcal step Depending on the epitope and the antibody used, the amounts of recovered DNA may vary substantially. It is important to measure the DNA concentration properly, as the performance of subsequent library preparation is highly dependent on the proper amount of input DNA. For the antibodies listed in table 1, 1–3 µl of sample was sufficient to obtain reliable concentration measurements. crItIcal step It is necessary to evaluate the IP efficiency by real-time PCR of known genomic-positive and genomic-negative target regions. If such regions are not known a priori they can be assessed by sequencing, but it is generally advised to assure the quality of the IP before costly sequencing. More general guidelines for designing and performing quantitative PCR have been published previously28.

54| For the QA sample (see Steps 32 and 33), run ~400 ng on a 1.5% (wt/vol) agarose gel. The fragment size should be ~200 bp for sequencing (Fig. 5a). pause poInt Samples can be stored at − 80 °C for at least 1 year.

library preparation for solexa sequencing ● tIMInG 7 h55| Aliquot 2–10 ng of immunoprecipitated DNA into an 0.5-ml PCR tube (2 ng being the minimum, 10 ng being the optimum). crItIcal step This protocol describes the use of the NEBNext ChIP-seq sample prep master mix kit for generating Solexa sequencing libraries. Although we have used the protocol described here successfully with amounts as low as 2 ng of DNA, it is generally advisable to use 5–10 ng of DNA to minimize PCR and sequencing artifacts.

56| Add sterile water to a final volume of 44 µl. Add 5 µl of NEBNext end-repair reaction buffer, and 1 µl of NEBNext end-repair enzyme mix. Pulse-vortex the reaction, briefly centrifuge and incubate the mixture for 30 min at 20 °C in a thermal cycler. crItIcal step This incubation serves to blunt-end the immunoprecipitation-enriched DNA fragments using a combination of Klenow and T4 DNA polymerases; polynucleotide kinase (PNK) ensures that each fragment has a 5′ phosphate, which is needed for efficient adapter ligation.

57| Purify the DNA using QIAquick PCR purification columns according to the manufacturer’s recommendations, but avoid optional washes and extend the drying spin after the PE buffer (part of Qiagen kit) wash to 2 min. Next, incubate in 44–45 µl of EB buffer (part of the Qiagen kit) added to the middle of the column membrane for 3 min at room temperature and elute DNA into a 1.5-ml low-binding tube by 3 min of centrifugation. crItIcal step Thorough removal of ethanol in the PE buffer is essential. Note that the next step requires 44 µl of end-repaired DNA, but QIAquick PCR purification columns have a small but consistent loss of 1–2 µl in the final elution. pause poInt Eluate can be stored at − 20 °C for at least 1 year.

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58| Add 5 µl of 10× NEBNext dA-tailing reaction buffer and 1 µl of Klenow fragment (3′→5′ exo–) to the 44 µl of end-repaired DNA.

59| Incubate the 50-µl reaction, after pulse-vortexing and spinning down, for 30 min at 37 °C. crItIcal step This incubation serves to add terminal adenines to the 3′ ends, which are required for adapter ligation.

60| Purify the DNA using Qiagen MinElute PCR purification columns according to the manufacturer’s recommendations, but avoid optional washes and extend the drying spin after the PE buffer wash to 2 min. Next, incubate DNA in 19–20 µl of EB buffer added to the middle of the column membrane for 3 min at room temperature and elute it into a 1.5-ml low-binding tube by 3 min of centrifugation. crItIcal step Thorough removal of ethanol in the PE buffer is essential. Note that the next step requires 19 µl of end-repaired DNA, but Qiagen MinElute PCR purification columns have a small but consistent loss of 1–2 µl in the final elution. pause poInt Eluate can be stored at − 20 °C for at least 1 year.

61| Transfer 19 µl of dA-tailed DNA to 0.5-µl PCR tubes.

62| Add the following components to the PCR tubes.

component amount (ml) Final

NEBNext quick ligation reaction buffer, 5× 6 1×

PE (paired end) Illumina adapters (1.5 µM; 1/10 dilution of manufacturer’s 15 µM stock)

1 50 nM

Quick T4 DNA ligase 4 8,000 units

63| Incubate the 30-µl reaction, after pulse-vortexing and spinning down, for 15 min at 20 °C in a thermal cycler. crItIcal step This incubation serves to T/A-ligate forked Illumina adapters to the dA-tailed DNA via the 3′ adenine overhang. Illumina adapters may need to be diluted to the appropriate concentration (see manufacturer’s recommendations). Other Illumina adapters or indexed adapters for multiplexing may be substituted according to the downstream application requirements. Although we perform single-end sequencing, paired-end adapters serve well; this ChIP-seq protocol yields nondirectional sequencing information.

64| Purify the DNA using Qiagen MinElute PCR purification columns according to the manufacturer’s recommendations, but avoid optional washes and extend the drying spin after the PE buffer wash to 2 min. Incubate the DNA in 20 µl of EB buffer added to the middle of the column membrane for 3 min at room temperature and elute it into a 1.5-ml low-binding tube by 3 min of centrifugation. crItIcal step Thorough removal of ethanol in the PE buffer is essential.

pause poInt Eluate can be stored at − 20 °C for at least 1 year.

65| Add 4 µl of 6× loading dye for agarose gel electrophoresis.

66| Prepare a 2% (wt/vol) agarose gel, prestained with GelGreen or another DNA stain excitable by blue light.! cautIon GelGreen is toxic and gloves should be always worn while working with it.

67| Load the sample into a small well just large enough to hold the entire sample and run the sample next to but separated by empty wells on either side from a 100-bp DNA ladder. Run the gel until the 200-bp and 300-bp ladder markers are well separated. crItIcal step Slower-running gels usually allow for cleaner fragment separation. Progress can be monitored on a blue-light box. The use of blue light rather than a UV light table is highly recommended to prevent DNA damage. The use of Invitrogen E-Gels is possible, but not necessary.

68| Align a clean straight edge ruler, using the markers to either side of the sample, so that the edge is just below where the bulk of the adapter-ligated DNA fragments should be; punch out a gel slice using SafeXtractor 100 tips (Fig. 5a). crItIcal step The amount of sample DNA will be too low to visualize directly. The point of cutting for size selection can be calculated as the average shearing size, which was estimated in Step 54, plus ~40–50 bases to account for the ligated adapters. It may be desirable to punch out backup slices slightly below and above the primary cut.

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69| Transfer the gel slice to a low-binding tube and measure the weight of the gel slice. pause poInt Gel slices can be stored at − 20 °C for at least 1 year.

70| Purify the DNA using QIAquick Gel Purification columns according to the manufacturer’s recommendations, but melt the gel slice at room temperature, avoid optional washes and extend the drying spin after the PE wash to 2 min. Next, incubate the DNA in 36–37 µl of EB buffer added to the middle of the column membrane for 3 min at room temperature and elute it into a 1.5-ml low-binding tube by a 3-min centrifugation. crItIcal step Thorough removal of ethanol in the PE buffer is essential. Note that the next step requires 36 µl of size-selected DNA, but QIAquick Gel purification columns have a small but consistent loss of 1–2 µl in the final elution. Melting the gel slice in QG buffer (included in the Qiagen kit) at room temperature, rather than at higher temperatures, is intended to avoid melting apart small, T/A-rich fragments32. pause poInt Eluate can be stored at − 20 °C for at least 1 year.

71| Transfer 36 µl of adapter-ligated and size-selected DNA to 0.5-ml thin-walled PCR tubes.

72| Add the following components to the PCR tubes; pulse-vortex and spin down the tubes.

component amount (ml) Final

Phusion high-fidelity buffer, 5× 10 1×

dNTP (2.5 mM each) 1.5 75 µM each

Illumina PE 1.0 PCR primer 1 0.5 µM

Illumina PE 2.0 PCR primer 1 0.5 µM

Phusion high-fidelity DNA polymerase 0.5 1 unit

73| Run the 50-µl PCR in a thermal cycler according to the following program:

step number Denature anneal extend Final

1 98 °C for 0:40

2–19 98 °C for 0:10 65 °C for 0:30 72 °C for 0:30

20 72 °C for 5:00 4 °C for

crItIcal step The number of PCR cycles can be varied. If you are starting with large amounts of immunoprecipitated material, as few as 15 cycles may be sufficient. It is generally advisable to use the fewest number of cycles possible to limit PCR artifacts. pause poInt PCR can be stored at − 20 °C after run completion for at least 1 year.

74| Add 10 µl of 6× loading dye for agarose gel electrophoresis.

75| Prepare a 2% (wt/vol) agarose gel, prestained with GelGreen or another DNA stain excitable by blue light.! cautIon GelGreen is toxic and gloves should be always worn while working with it.

76| Load the sample into a well just large enough to hold the entire sample and run the sample next to but separated by empty wells on either side from a 100-bp DNA ladder. Run gel until the 200-bp and 300-bp ladder markers are well separated. crItIcal step Slower-running gels usually allow for cleaner fragment separation. It is essential to run the gel far enough to achieve clean separation of the main PCR product and unincorporated primers and PCR artifacts such as primer dimers (which may run as high as 100–150 bp). Progress can be monitored on a blue-light box. The use of blue light rather than a UV light table is highly recommended to prevent DNA damage.

77| On a blue-light table, cut out the PCR band with a clean scalpel or razor blade, transfer the gel slice to 1.5- or 2-ml microcentrifuge tubes and measure the weight of the gel slice (Fig. 5a). pause poInt Gel slices can be stored at − 20 °C for at least 1 year.

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78| Purify the DNA using QIAquick Gel purification columns according to the manufacturer’s recommendations, but melt the gel slice at room temperature, avoid optional washes and extend the drying spin after the PE wash to 2 min. Incubate the DNA in 15 µl of EB buffer added to the middle of the column membrane for 3 min at room temperature and elute it into a 1.5-ml low-binding tube by a 3-min centrifugation. crItIcal step Thorough removal of the ethanol in the PE buffer is essential. Melting the gel slice in QG buffer at room temperature, rather than at higher temperatures, is intended to avoid melting-apart of small, T/A-rich fragments. The eluate is the amplified and size-selected library. pause poInt Eluate can be stored at − 20 °C for at least 1 year.

79| Measure the concentration of the purified DNA on a Qubit fluorometer.

80| Quality-assess the concentration and size distribution of amplified DNA fragments on an Agilent Bioanalyzer (Fig. 5b). crItIcal step For optimal Solexa sequencing performance, a sharp single peak is desired. Although a narrow size distribution allows for good cluster resolution during the Solexa flow cell imaging, secondary peaks are to be avoided and may be indicative of ineffective separation of PCR artifacts. Note that even low peaks of smaller DNA fragments are problematic, as their molar proportion may be high.

81| Submit the sample to clustering and Illumina Solexa sequencing per the manufacturer’s recommendations. If multiplexed samples are to be submitted, ensure that their DNA fragment sizes are closely matched, and mix them in an equimolar ratio.

? trouBlesHootInGTroubleshooting advice can be found in table 2.

taBle 2 | Troubleshooting table.

step problem possible reason solution

EQUIPMENT SETUP

FSC signal is noisy

Light scattering generated by the jet as a result of piezo oscillations and droplet generation is reaching the detector (photodiode). Check if events trigger acquisition in the absence of sample or with the sample valve closed

Adjust the position of the FSC collection lenses assembly. The FSC obscuration bar in the MoFlo must be tilted to let in as much particle-scattered light as possible in the forward direction. There is a tradeoff, however, as this might also increase direct laser and unwanted scatter background. Find an illumination position that allows optimal particle illumination but minimizes background noise. This position on the jet is found near the nozzle tip. Also decreasing the amplitude of the droplet generation or using a slightly suboptimal frequency can reduce the droplet generation scatter noise at the interrogation point

11 Low recovery of nuclei

Loss of sample because of the inefficient release of nuclei from the specimen

Increase the number of douncing and syringing steps

Broken and deformed nuclei

Ruptured nuclear integrity leads to release of chromatin from the nuclei

Decrease the number of douncing and syringing steps. Decrease the gauge of the needles

23 Poor resolution of positive and negative nuclei

Inefficient washing of nuclei; antibody conjugates remain in solution

Washing the nuclei in PBTB solution will reduce the amount of unbound dye. A single wash is usually sufficient to obtain a good staining for positive nuclei with little background, but the washing step may be repeated to obtain optimal separation

24 Fluidic instability Improper filtering of the sample before sort; air or dirt in the nozzle tip; obstructed sample lines

Remove the sample tube and filter the nuclei suspension through a 70-µm cell strainer or mesh. Allow some back flush through the sample line. Clean the sample lines by running 0.2-µm filtered BD FACSRinse solution or BD FACS clean solution. Checking for variations in the events rate can help you anticipate obstructions in the sample line and nozzle blockage. Remove the sample tube and resuspend nuclei halfway during long sorts. Periodically inspect the quality of the sort deflections and the stability of the break-off point, as they are good indicators of problems with fluidic instability

(continued)

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● tIMInGSteps 1–11, extraction of nuclei: ~1 hSteps 12–15, cell type–specific nuclear staining: 2.5 h (optional steps)Steps 16–26, sorting of labeled cell type–specific nuclei: variable (4–12 h); the time spent sorting can vary dramatically depending on the quality of the preparation, the percentage of desired nuclei in the sample, fluorescence intensity, the amount that needs to be recovered, and the sorter used (allowed sample frequency and dim fluorescence sensitivity)Steps 27–33, lysis of nuclei and shearing of the chromatin: ~1 hSteps 34–39, preabsorption and immunocomplex formation: 1 d (1.5 h for sample preparation, 1 h for preincubation, over-night for incubation)Steps 40–46, purification of immunocomplexes: 1 d (3 h for incubation, 2 h for wash steps, 30 min for RNase digest and overnight for proteinase K digestion)Steps 47–54, cross-link reversal and DNA purification: 8 h (6 h for incubation, 30 min for extraction, 1.5 h for precipitation)Steps 55–81, library preparation for Solexa sequencing: 7 h

antIcIpateD resultspurification of cell type–specific nuclei (steps 1–26)The successful purification of cell type–specific nuclei depends primarily on three parameters. First, it requires a clean nuclear preparation, consisting of single, intact, fixed nuclei (Fig. 2b). In general, a good nuclear preparation should be devoid of contaminant intact cell clumps and other large particulate matter and should not contain subnuclear debris, such as free floating chromatin (Fig. 2d, ‘overshearing’). Second, a successful purification should have a good fluorescent signal of the cell type–specific labeled nuclei with low background fluorescence of the unlabeled nuclei. Although the specificity of the fluorescent signal can be assessed using regular fluorescence microscopy (Fig. 2), it is of pivotal importance that the sorter can cleanly distinguish the two populations. FSC and fluorescence (Alexa488-log) plots should show well-separated clusters of positive, fluorescent nuclei and unlabeled nuclei as shown in Figure 4. Finally, correct instrument setup is crucial—follow the manufacturers’ instructions to determine the correct droplet break-off time (Fig. 3).

In our hands, a good nuclear preparation and careful FACS setup and post-sort handling resulted in cell type–specific samples with a purity > 97% (Fig. 2c) and recoveries varying between 40% and 60% from a single sort. Although high recovery is not an indicator for the sample quality or purity, high recovery reduces the sorting time needed to obtain a given amount of nuclei and is an indicator of optimal instrument setup (for example, drop delay).

chromatin shearing (steps 27–33)The optimal shearing size of chromatin depends on the downstream application. For ChIP-seq, the fragment length should be ~200 bp (Fig. 5a), ChIP-on-chip usually requires sizes of ~500 bp. Typically, optimally sheared chromatin appears as a smear on an agarose gel that is centered around the target size (e.g., 200–250 bp for ChIP-seq). Oversheared chromatin will have smaller-than-optimal fragments (e.g., < 200 bp for ChIP-seq) and should be discarded.

taBle 2 | Troubleshooting table (continued).

step problem possible reason solution

26 Purity < 95% Suboptimal instrument alignment; FSC threshold set too high; fluidic instability; drop delay shifts

Decrease the threshold on FSC to allow detection of nuclei low-end scatter. Acquire Flow-Check fluorospheres to confirm that the alignment is optimal. Correct if necessary. See TROUBLESHOOTING advice in Step 24 for fluidic instability issues. Calculate the instrument drop delay

33 Sheared chromatin fragments are too large

Inefficient shearing of the chromatin during sonication

Optimize the shearing conditions on unsorted material using a fixed amount of chromatin (nuclei). Too much chromatin can result in inefficient shearing. Alternatively, increase the number and/or length of sonication cycles

Sheared chromatin fragments are too small

Excessive shearing of the chromatin during sonication

Optimize the shearing conditions on unsorted material using a fixed amount of chromatin (nuclei). Too little chromatin can result in excessive shearing. Alternatively, decrease the number and/or length of sonication cycles

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chromatin Ip and library preparation (steps 34–81)By using Drosophila embryos, a successful ChIP on histone modifications (for example, H3K4me1, H3K79me3, H3K36me3 and H3K27ac) should recover 10–50 ng of DNA from 3 to 10 µg of cross-linked input chromatin (table 1). In contrast, ChIP on TFs and RNA Pol II yields 10–15 ng of immunoprecipitated DNA, starting with 10–15 µg of cross-linked input chromatin (table 1). We have previously described a more detailed protocol on how to assess good ChIP performance28.

For optimal Solexa performance in terms of cluster generation and resolution, it is advisable to generate a library of narrow fragment size distributions. For quality assurance, we first determine the concentration of our PCR-amplified and gel-purified sequencing libraries using a Qubit fluorometer. A successful PCR amplification should yield DNA concentrations in excess of 2 ng µl − 1, and we routinely obtain concentrations of 20–50 ng µl − 1. To further determine the fragment size distribution and mean fragment size, we advise running small library aliquots on an Agilent 2100 Bioanalyzer using DNA 1000 or high- sensitivity DNA chips. The library should show a single peak at the expected fragment size (secondary peaks may indicate primer dimers or aberrant PCR products) and a narrow size distribution (a large spread of fragment sizes can affect the sequencer’s cluster identification and separation ability) as exemplified in Figure 5b.

acknoWleDGMents We thank E.H. Gustafson for fly work, and J. Erceg for sharing staged, sorted nuclei. We are very grateful to all members of the Furlong Laboratory, J. Mueller, C. Margulies and A.G. Ladurner for helpful discussions. This work was supported by grants to E.E.M.F. from ERASysBio (Mod Heart) and the Human Frontiers Science Organization, and by a long-term fellowship to R.P.Z. from the International Human Frontiers Science Program Organization. S.B. was funded by the European Molecular Biology Laboratory Interdisciplinary Postdoctoral Programme.

autHor contrIButIons S.B., R.P.Z. and E.E.M.F. designed the study. A.P.-G. and A.R. conducted the FACS experiments. S.B. and R.P.Z. conducted all other experiments. S.B., R.P.Z., A.P.-G., A.R., A.-C.G. and E.E.M.F. wrote the manuscript.

coMpetInG FInancIal Interests The authors declare no competing financial interests.

Published online at http://www.nature.com/doi:10.1038/nprot.2012.049. Reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

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Cell type–specific chromatin immunoprecipitation from multicellular complex samples using BiTS-ChIP