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RNAExpressionProfilingfromFACS-IsolatedCellsoftheDrosophilaIntestine

ARTICLEinCURRENTPROTOCOLSINSTEMCELLBIOLOGY·NOVEMBER2013

DOI:10.1002/9780470151808.sc02f02s27·Source:PubMed

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UNIT 2F.2RNA Expression Profiling fromFACS-Isolated Cells of the DrosophilaIntestine

Devanjali Dutta,1 Jinyi Xiang,1 and Bruce A. Edgar1

1DKFZ-ZMBH Alliance, University of Heidelberg, Heidelberg, Germany

ABSTRACT

This unit describes a protocol for the isolation of Drosophila intestinal cell populationsfor the purpose of cell type–specific transcriptome profiling. A method to select a celltype of interest labeled with green or yellow fluorescent protein (GFP, YFP) by makinguse of the GAL4-UAS bipartite system and fluorescent-activated cell sorting (FACS)is presented. Total RNA is isolated from the sorted cells and linear RNA amplificationis used to obtain sufficient amounts of high-quality RNA for analysis by microarray,RT-PCR, or RNA sequencing. This method will be useful for quantitative transcriptomecomparison across intestinal cell types under normal and various experimental conditions.Curr. Protoc. Stem Cell Biol. 27:2F.2.1-2F.2.12. C© 2013 by John Wiley & Sons, Inc.

Keywords: Drosophila intestinal stem cells � fluorescent-activated cell sorting (FACS)� cell type–specific RNA isolation � transcriptome profiling/RNASeq � enterocyte

INTRODUCTION

The epithelial cell lining of the gastrointestinal tract in Drosophila melanogaster(Drosophila/fruitfly) is maintained by a continuous supply of cells, which arise by the dif-ferentiation of multipotent progenitors that originate from intestinal stem cells (Micchelliand Perrimon, 2005; Ohlstein and Spradling, 2007). Intestinal stem cells (ISCs) prolifer-ate throughout the life of the adults, replacing themselves and generating transient cellscalled enteroblasts (EBs), which differentiate into enterocytes (ECs) or enteroendocrinecells (EEs). Transcription factor Escargot (esg) is a common marker for both ISCs andEBs. In addition to these cell types, the midgut region of the intestine is ensheathed bytwo layers of visceral muscle (VM) cells and supplied with oxygen by the trachea. Theproper regulation of intestinal stem cell maintenance, proliferation, and differentiationis critical for maintaining gut homeostasis (Jiang et al., 2009). As the understanding ofstem cell development and function in vivo becomes more sophisticated, it has becomeimportant to profile the various intestinal cell types at the transcriptome level. Althoughexpression studies have been performed on the whole midguts of adult Drosophila(Buchon et al., 2009; Jiang et al., 2011; Osman et al., 2012), cell type–specific tran-scriptome profiling of the Drosophila midgut cell types has not yet been undertaken.A protocol that can be useful for quantitative comparisons of gene expression acrossthe different intestinal cell populations of the Drosophila midgut under physiologicalor any experimental condition of overexpression or knockdown is presented. This unitbegins with a method to dissociate and purify various intestinal cell types by enzymaticdissociation and FACS (see Basic Protocol 1), followed by a protocol to isolate andamplify RNA linearly (see Basic Protocol 2), and then by a protocol to stain the sortedcells to observe their morphology (see Basic Protocol 3). The amplified RNA can beused subsequently for gene expression profiling experiments. A schematic overview ofthe major steps in the procedure is shown in Figure 2F.2.1A.

Current Protocols in Stem Cell Biology 2F.2.1-2F.2.12, November 2013Published online November 2013 in Wiley Online Library (wileyonlinelibrary.com).DOI: 10.1002/9780470151808.sc02f02s27Copyright C© 2013 John Wiley & Sons, Inc.

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4. total RNA isolation fromFACS-sorted cells

8. immunofluorescence stainingof FACS-sorted cells

3. FACS sort GFP+, PI-cells

living

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1. dissect midguts ISC-DI-GFP ISC-DI-GFP

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6. RNA sequencing of aRNAon Illumina GAllX platform

Figure 2F.2.1 FACS sorting of Drosophila intestinal cell populations for obtaining pure cell populations. (A) Afterdissection of midgut, tissue was disrupted by enzymatic treatment and the cell suspension was FACS sorted. Cellswere gated based on GFP intensity and cell size. RNA was isolated from the FACS-sorted cells followed by tworounds of cDNA synthesis and in-vitro transcription for RNA amplification. Amplified RNA was subjected to qRT-PCRanalysis or Illumina GAIIX 72-bp mRNA sequencing. Immunofluorescence staining of sorted cells was performed tocheck for morphological changes. Genotypes used were esg-Gal4ts, UAS-GFP. (B) Detailed methodology of FACSsorting of cell populations. Cells were first gated for forward scatter (FSC-A) and side scatter (SSC-A); aggregatesand doublets are then removed by selecting singlets using dot-plots of SSC-W versus SSC-H and FSC-W versusFSC-H. Finally, propidium iodide–negative (living) and GFP-positive cells were sorted out.

NOTE: The following protocols are performed under Class II biohazard conditions.

NOTE: All reagents and equipment should be sterile and RNAse-free conditions shouldbe maintained.

BASICPROTOCOL 1

ISOLATION AND PURIFICATION OF DROSOPHILA INTESTINAL CELLSBY FLUORESCENT-ACTIVATED CELL SORTING (FACS)

This protocol describes tissue preparation and isolation of the intestinal cell typesby FACS. The following adult fly Gal4 lines driving UAS-GFP or UAS-YFP

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expression—esg-Gal4 (ISCs +EBs), Dl-Gal4 (ISCs), Su(H)-Gal4 (EBs), Myo1A-Gal4(ECs), How-Gal4 (visceral muscle), and Rab3-YFP (EEs)—should be used to select thedifferent cell populations. Pros-Gal4 driving GFP expression does not mark the enteroen-docrine cells specifically and thus its use is not recommended. All of the dissection toolsand dishes should be wiped with RNaseZap from Ambion prior to use. Keep dishesplaced on ice throughout.

Materials

Transgenic adult fly Gal4 lines driving GFP or YFP expression: esg-Gal4 (ISCs+EBs), Dl-Gal4 (ISCs), Su(H)-Gal4 (EBs), Myo1A-Gal4 (ECs), How-Gal4(visceral muscle) and Rab3-YFP (EEs)

10× PBS-DEPC (see recipe)RNaseZap (Ambion) or 70% ethanolElastase (see recipe) or collagenase type IV (see recipe)1 mg/ml propidium iodide10% bleachDEPC-treated water (see recipe)Wild-type control wIII8 flies

Dissection dishesDissecting tools: scissors, fine forcepsDissecting microscope1.5-ml centrifuge tubes27°C heating block with shakingRefrigerated centrifuge25- and 70-µm filter unitsFACSAria II cell sorter (Becton Dickinson)FACS tubes with filter topsBD FACSDiva v6.1.1 or similar software

1. Use CO2-anesthetized adult female flies, as it is preferable to use either female ormale midguts as mixing both could skew the transcriptome profiles due to gender-specific variations.

Use of female flies is recommended for obtaining more cells of each cell population dueto the ease of dissection and larger size.

2. Remove the head, wings, and legs of the fly. Transfer fly into a well of a dissectiondish containing 2 ml of 1× PBS-DEPC to start dissections. Use a pair of fineforceps to gently hold the fly between the thorax and abdomen and gently squeezethe abdomen to push the midgut out from the anterior end. With another pair offorceps, pull out the gut slowly but steadily. Pull carefully to obtain the undamagedgut, cut off the proventriculus, crop, and anus. If malphigian tubules are pulled outas well, dissect them away from the gut.

A total of 100 to 150 midguts will be needed for the protocols, however, dissecting 10 to15 flies at a time is recommended to avoid exposing the midgut tissue to room temperaturefor long periods of time.

3. Use forceps to immediately transfer all of the guts into a 1.5-ml microcentrifugetube containing PBS-DEPC (�400 µl), placed on ice. Keep total dissection time<2 hr.

To avoid variations in gene expression patterns as a function of temperature, the dissectedmidguts should be maintained at 4°C until the dissociation step.

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Figure 2F.2.2 Cell type–specific GAL4 drivers and FACS profiles of Drosophila intestinal cell populations. Variousgenotypes were used to isolate different cell populations. FACS profiles of various cell types are shown. (A, A′) esg-Gal4ts, UAS-GFP; (B, B′) Myo1A-Gal4ts, UAS-GFP; (C, C′)- How-Gal4ts, UAS-GFP; (D, D′) Dl-Gal4ts, UAS-GFP; (E, E′)Su(H)-Gal4ts, UAS-GFP; (F, F′) Rab3-YFP; (G, G′, G′ ′) Immuno fluorescence staining for the intestinal stem cells showedno effect of FACS on cell morphology with cells expressing marker Delta and GFP respectively. Genotypes used wereesg-Gal4ts, UAS-GFP. Dissociated FACS-sorted cells are highly pure populations of cells that retain their morphologicalfeatures and marker expression.

4. Collect �150 midguts (for ISCs, EBs, EEs) and 100 midguts (for esg+ cells,ECs, and VM) in a 1.5-ml centrifuge tube and digest with 100 µl elastase solution(1 mg/ml final concentration).

More midguts are dissected for less abundant cell types like ISCs, EEs, and EBs in orderto obtain enough cells of the particular cell type after sorting.

Collagenase type IV can also be used for isolating the esg+ cells depending on avail-ability; however, the efficiency of elastase was found to be higher and more livingcells could be obtained, particularly for flies expressing membrane GFP (mGFP). The

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efficacy of collagenase for ECs and the VM was not determined. Dissect the sampleswithin 2 hr. The efficacy of elastase may vary with batch and storage thus needs to bechecked regularly.

5. Incubate tubes for 1 hr at 27°C in a heating block with shaking at 34 × g. Agitateevery 15 min by pipetting up and down at least 30 to 40 times to fully dissociate thetissue.

6. Centrifuge dissociated cells for 20 min at 300 × g, 4°C and re-suspend pellet in500 µl fresh PBS-DEPC for sorting. Add 1 µl of 1 mg/ml propidium iodide.

7. Filter dissociated cells using 25-µm (for ISCs, EBs, esg+ cells, and EEs) and 70-µm(for ECs and VM) filters to remove clumps, which might otherwise block the nozzleof the FACS sorter. Maintain cells throughout at 4°C.

The time and agitation for dissociation to obtain ECs and visceral muscle should beincreased in case clumps are still seen even after 1 hr.

8. Pre-treat BD FACSAria II cell sorter with 10% bleach for 180 sec followed bywashing three times, 180 sec each, with DEPC water. Sort samples at 4°C and23 psi pressure using the 85-µm nozzle for the ISCs, EBs, ESG+ cells, and EEs.Use100-µm sized nozzle for sorting ECs and visceral muscle cells.

Use BD FACSDiva v6.1.1 or similar software for collection, storage, and analysis of thedigital data.

9. To account for auto fluorescence, set gates using midguts from wIII8 flies. GFP-positive cells were sorted based on two criteria—fluorescence intensity and size ofcells. Gate cells initially using forward scatter (FSC-A) and side scatter (SSC-A).Remove clumps and doublets by gating singlets in two linear scale dot-plots ofSSC-W versus SSC-H and FSC-W and FSC-H (Fig. 2F.2.1B).

10. Sort out propidium iodide-negative (living), GFP-positive cells. Perform the proce-dure three times to obtain biological triplicates. Enrich the cell type fractions usingFACS Aria cell sorter (see equipment setup) and cell type–specific marker driversexpressing green and yellow fluorescent protein (GFP/YFP) (Fig. 2F.2.2A-F, A′-F′).

After cell sorting, immediately proceed to RNA isolation. Storage of sorted cells inextraction buffer even at −80°C decreases RNA yield.

For the purpose of culturing or immunostaining of sorted cells, sorting of cells directlyinto Schneider’s medium is recommended.

BASICPROTOCOL 2

RNA ISOLATION AND AMPLIFICATION

For the purpose of RNA isolation, one genotype should be dissected at a time and at leastthree biological replicates should be prepared for each cell type. It is recommended thatthe same numbers of cells are sorted out and the same amount of RNA is amplified foreach cell type or for comparing experimental conditions with controls. Amplification ofRNA is necessary for smaller cells like ISCs, EBs, and EEs due to their low yield of RNA,whereas RNA from esg+ cells and ECs can be used directly for gene expression studies.However, as linear amplification introduces bias towards the longer transcripts and mightskew analysis, it is thus not advisable to compare amplified and unamplified samples.Amplifying RNA from all the cell types being studied or compared is recommended.

NOTE: RNaseZap (Ambion) is used throughout to keep RNase-free conditions.

Materials

Sorted cells (see Basic Protocol 1)Arcturus PicoPure RNA isolation kit (Applied Biosystems)

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70% RNase-free ethanolArcturus RiboAmp HS PLUS RNA amplification kit (Applied Biosystems)SuperScript III reverse transcriptase (Invitrogen, cat. no. 18080-44)

1.5-ml microcentrifuge tubesParafilm42°C water bathRefrigerated centrifugeThermal cyclerQubit 2.0 Fluorometer (Invitrogen)

1. Use the Arcturus PicoPure RNA isolation kit for RNA isolation with the followingmodifications—sort 2000 cells each into 1.5-ml microcentrifuge tubes containing500 µl of RNA extraction buffer (place immediately on ice).

2. Seal sorted cells in microcentrifuge tubes with Parafilm and incubate 1 hr in a 42°Cwater bath.

Incubation times can vary between 45 min and 1 hr but not less.

3. After incubation, centrifuge cells 4 min at 3000 × g, 4°C. Transfer supernatantinto a new microcentrifuge tube and add 500 µl of 70% RNase-free ethanol, andmix well. Follow the Arcturus PicoPure RNA isolation kit per the manufacturer’sinstructions, adding the extraction buffer-alcohol mixes in increments of 300 µl tothe RNA extraction columns.

4. Elute the RNA with 12 µl of elution buffer and store at −80°C or proceed to amplifi-cation.

5. Program the thermal cycler as per Arcturus RiboAmp HS PLUS RNA amplificationkit instructions (Tables 2F.2.1 and 2F.2.2).

Table 2F.2.1 RiboAmp HS PLUS Thermal Cycler Program Round Onea

Temperature (°C) Time

First-strand synthesis 65 5 min

4 Hold

42 1 hr

4 Hold

37 30 min

95 5 min

4 Hold

Second-strand synthesis 95 2 min

4 Hold

25 10 min

37 30 min

70 5 min

4 Hold

IVT 42 6 hr

4 Hold (optional overnight hold)

37

15

4 Hold

aFrom Applied Biosystems.

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Table 2F.2.2 RiboAmp HS PLUS Thermal Cycler Program Round Twoa

Temperature (°C) Time

First-strand synthesis 65 5 min

4 Hold

25 1 hr

37 30 min

4 5 min

Second-strand synthesis 95 2 min

4 Hold

37 30 min

70 5 min

4 Hold

IVT 42 6 hr

4 Hold (optional overnight hold)

37

15

4 Hold

aFrom Applied Biosystems. The program is not intended for automatic progression from one time and temperature setto another. The program lists a 4°–8°C hold after each incubation or incubation cycle when it is necessary to remove thereactions from the thermal cycler to add reagents. After the addition of reagents, place the sample back into the thermalcycler and resume the program. Using a thermal cycler with a heated lid is important. The heated lid ensures propertemperature distribution within the reaction tube and prevents evaporative condensation that alters the reaction mixtureconcentrations. The 4°–8°C steps in the thermal cycler program allow for buffer and reagent addition and mixing steps atcertain points during the amplification process and are not intended for indefinite hold unless noted (Applied Biosystems).

The amplification cycle steps should be saved as a single program with breaks at 4°C. Themachine should not be switched off any time in between the procedure.

6. Quantify RNA using the Qubit 2.0 Fluorometer. Use 2 ng of RNA for amplification.Amplify the RNA using Arcturus RiboAmp HS PLUS RNA amplification kit.

7. Use amplified RNA directly for RT-PCR, microarray, or RNA sequencing.

BASICPROTOCOL 3

IMMUNOFLUORESCENCE STAINING OF SORTED STEM CELLS

The staining protocol for Drosophila intestinal stem cells is adapted from the protocolused for the staining of germline cells, as described in UNIT 2E.3. It has been concluded thatthe FACS sorting procedure does not cause any detectable modification of ISC properties(Fig. 2F.2.2G-G′′). Positively charged slides were used and the procedure was performedin a humidified chamber (wet tissues in petri plate) and without exposure to directlight. Midguts were stained with mouse monoclonal anti-Delta (1:100, rabbit polyclonalanti-GFP, 1:1000, Invitrogen) and DNA was stained with Hoechst 33258 (Invitrogen).

Materials

Sorted cells (see Basic Protocol 1)Schneider’s medium (PromoCell)Fetal bovine serum (FBS)Formaldehyde0.15% PBST (see recipe)5% NGS in PBST (see recipe)

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Antibodies: mouse monoclonal anti-Delta (1:100), rabbit polyclonal anti-GFP(1:1000) (Invitrogen)

0.1% DAPIVectashield mounting medium (Vector)

1.5-ml microcentrifuge tubesRefrigerated centrifugePositively charged slidesPetri platesCoverslipsMicroscope

1. Sort out 10,000 cells in a 1.5-ml microcentrifuge tube containing 500 µl of Schnei-der’s medium with 10% FBS (S-FBS).

2. Centrifuge 5 min at 500 × g, 4°C and resuspend in 100 µl of S-FBS.

3. Pipet suspension onto a positively charged slide in a humidified chamber (wet tissuesin a petri plate) and allow cells to settle for 1.5 hr.

Avoid exposure to direct light during the staining procedure (cover chamber with atissue).

4. Fix cells by adding 500 µl of 1:3 (v/v) formaldehyde/Schneider’s medium onto theslide and allow cells to stand for 30 min in the humidified chamber (away fromdirect light) at room temperature.

5. Hold the slide at a 45° angle and wash at least five times with 1 ml of 0.15% PBST.

6. Block by adding 500 µl of 5% NGS in PBST onto the slide for 30 min. Wash offblocking solution by slowly pipetting 1 ml of 0.15% PBST onto the slide to drainoff excess blocking solution. Wash at least three times.

7. Add 500 µl primary antibody in PBST with 5% NGS onto the slide and incubate for1.5 hr at room temperature. Wash off excess primary antibody five to six times with1 ml of 0.15% PBST.

8. Add 500 µl secondary antibody in PBST with 5% NGS and incubate for 1 hr. Rinsewith PBST.

9. Add 0.1% DAPI (1:200 in 1× PBS) onto the slide and let stand for 5 min at roomtemperature. After incubation, hold slide at a 45° angle and wash five to six timeswith 1 ml PBST.

10. Add 25 µl mounting medium (Vectashield) and add coverslip onto the slide. Observeunder a microscope.

Round GFP-positive cells can be observed, which represent the progenitor cells. Deltaantibody marks the intestinal stem cells specifically and co-localizes with GFP-positivecells.

REAGENTS AND SOLUTIONSFor culture recipes and steps, use sterile tissue culture–grade water. For other purposes,use deionized, distilled water or equivalent in recipes and protocol steps. For suppliers, seeSUPPLIERS APPENDIX.

Collagenase solution, 1% (w/v)

Dissolve lyophilized collagenase type IV (Sigma, cat. no. C8051) in RNase-free PBSto a concentration of 50 mg/ml. Filter through a 0.22-µm filter. Prepare 1-ml aliquots

continued

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and store up to 3 months at −20°C. Thaw stock solution from freezer immediatelybefore use and dilute into 1× PBS-DEPC (see recipe) to a final concentration of10 mg/ml.

DEPC-treated water and PBS-DEPC, 0.1% (v/v)

Add 1 ml of fresh diethyl pyrocarbonate (DEPC) to 1 liter of distilled water or 1×PBS (10× PBS, Ambion). Shake well to disperse the DEPC through the solutionand leave overnight on a shaker at room temperature (room temperature, 22° to25°C). Autoclave at 15 p.s.i. on liquid cycle for 20 min to inactivate the remainingDEPC. The solution remains stable for many months in RNase-free conditions atroom temperature.

Elastase solution, 0.4% (w/v)

Reconstitute elastase (Sigma, cat. no. E0258) in cell dissociation buffer at a concen-tration of 4 mg/ml and store at −20°C. Avoid multiple freeze-thaws.

NGS in PBST, 5% (v/v)

Add 5 ml NGS (Sigma) to 100 ml PBST

PBST, 0.15% (v/v)

Mix 0.15 ml Triton X-100 (Merck) in 100 ml of 1× PBS

COMMENTARY

Background InformationWhile isolation of frequently occurring

cell types is relatively easy and enables tran-scriptome, proteome, and metabolome stud-ies; profiling of rare cell types still remainsa challenge. Several methods have been de-scribed for the isolation, purification, andprofiling of rare cell types and adult stemcells from various mammalian and Drosophilatissues. These include (1) 4-thiouracil (TU)tagging, e.g., from Drosophila larvae, em-bryos, and adults and other mammalian tis-sues as described by Miller et al. (2009);(2) RNA co-immunoprecipitation or mRNA-tagging, e.g., from muscles and sensory neu-ronal cells in C. elegans (Roy et al., 2002;Kunitomo et al., 2005); (3) isolation of nu-clei tagged in specific cell types (INTACT),e.g., from C. elegans muscle cells, from themesoderm of Drosophila embryos and neu-rons as described by Steiner et al. (2012);(4) fluorescent-activated cell sorting (FACS),e.g., from Drosophila neuronal stem cells andembryonal germline cells and mammalian tis-sues (Shigenobu et al., 2006; Berger et al.,2012; Pasut et al., 2012); and (5) microdis-section or mechanical purifications, e.g., forvarious plant tissues (Leonhardt et al., 2004;Schmid et al., 2012). While mechanical meth-ods and microdissection require the cells to bedetectable for isolation, TU-tagging is recom-mended for frequently occurring cells wheremodified 4-thiouracil can be incorporated into

newly synthesized RNA in cells express-ing uracil phosphoribosyltransferase (UPRT),thus enabling cell type–specific RNA isola-tion. The INTACT method involves affinitypurification of nuclei from specifically taggedcells and thus renders the isolation of wholecells unnecessary. It has the added advan-tage of allowing the study of chromatin fea-tures or epigenetic profiling of rare cell types.However, more optimization of the methodis needed. mRNA tagging involves taggingmRNA from a specific tissue by express-ing a FLAG-tagged poly-(A) binding protein(PABP) and separation of the mRNA of thespecific tissue by co-immunoprecipitation us-ing an anti-FLAG antibody (Yang et al., 2005).FACS facilitates isolation of rare cell typesfluorescently tagged with cell type–specificmarkers, from complex tissues. It is an ex-tremely sensitive method and allows isola-tion of cells with very low false positives,which makes it a powerful and straightfor-ward tool for cell type specific studies. Usingvarious reporters and the bipartite GAL4/UASsystem (Fischer et al., 1988; Brand and Per-rimon, 1993) several Drosophila cell typeshave been purified. Recently, female undif-ferentiated germ line cells from the adultDrosophila ovaries were isolated using FACS(UNIT 2E.3).

The Drosophila midgut has well charac-terized markers, with cell type–specific GAL4drivers available for all the different cell

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populations. The objective was to develop areproducible, fast, easy, and efficient methodto profile the various cell types with high accu-racy using the existing resources and withoutrequiring unusual equipment or technical spe-cialization. A combination of the GAL4/UASsystem, FACS, RNA isolation, and linear RNAamplification (using commercially availablekits) has been used to facilitate gene expres-sion profiling of the Drosophila intestinal celltypes. Described in this unit is a protocol thatcan be used to isolate the endodermally de-rived undifferentiated stem (ISCs), progenitorcells (EBs), the differentiated enterocytes andenteroendocrine cells (ECs and EEs), and themesodermally derived visceral muscle (VM)with high precision. With minor variations inthe protocol for each cell type, it can be usedto study the gene expression profile of anycell population in the Drosophila midgut un-der any experimental condition of knockdownor gene over expression. This makes it a pow-erful tool to study cell type–specific variationsin the Drosophila intestinal populations.

In addition to FACS, TU-tagging, INTACT,micro-dissection, and magnetic bead sortinghave been used to purify RNA from specificcell populations from tissues. TU-tagging,PABP CoIP, or the INTACT method could inprinciple be used for the fly gut instead ofthe FACS protocol, but optimization for usein the Drosophila intestine would be required.The yield after CoIP of RNA from stem cellsmight be problematic, making FACS a morefeasible method. While magnetic bead sortingis believed to be a milder method than FACSfor cell purification and might cause less celldeath (Zou et al., 2011; Iyer and Cox, 2010),with the FACS protocol described here, >90%propidium iodide–negative (living) cells hasbeen consistently observed, indicating that theconditions in which cell isolation is performedare not harsh and do not cause enormous celldamage or death.

Previously, Drosophila intestinal cells havebeen analyzed by FACS to determine the cellcycle phases of cells under various experimen-tal conditions (Amcheslavsky et al., 2011).However, the described dissociation protocoluses trypsin for dissociation, which is foundto be damaging for the cells as fewer livingcells were obtained subsequent to treatment(<60%). Also, due to the time required for thedescribed preparation, it would not be idealfor gene expression studies. Milder enzymes(collagenase, elastase) that are optimized forisolation of all cell types of the midgut, eithermembrane or nuclear GFP labeled, are used.

Immunofluorescent staining of FACS-sortedISCs showed that cells retained their morpho-logical characteristics and marker expressionafter sorting, and thus the FACS procedure ap-pears not to cause any major modification ofthe stem cell properties. This protocol mightalso be adapted for cell type–specific studies ofother cellular constituents such as chromatin,proteins, lipids, or various metabolites, pro-vided the end-analysis methods are sensitiveenough for the relatively small number of cells(2000) obtained. Since this FACS protocol iso-lates living cells, it could also conceivably beused to purify stem cells for in vitro culturestudies.

Using this protocol, transcriptome profilesof each Drosophila intestinal cell populationunder physiological conditions and also af-ter enteric infection by Pseudomonas ento-mophila (P.e) have been generated. Such adataset can be used to study the complexchanges occurring in the fly midgut upon in-fection in a cell type–specific manner. As theprofiling has been done under homeostaticconditions, the dataset can be used as a refer-ence for comparison with new transcriptomedata prepared from these cell types under newexperimental conditions, as long as the newdata were generated using the same protocol.The resource can be used to find novel celltype–specific markers and for developing newcell type–specific Gal4 “driver” lines. Usingthe cell type–specific transcriptome datasets,several novel markers for Drosophila intesti-nal stem cells have identified and verified (un-pub. observ.).

Critical Parameters andTroubleshooting

An equal number of cells should be sortedout for each cell type. All steps from dissec-tion of midguts up to RNA isolation shouldbe performed on the same day, since cell stor-age at −80°C in extraction buffer was foundto reduce the yield of RNA. To avoid delaysduring the procedure, all the settings for theFACS and PCR machines should be set before-hand. Pretreat the FACS machine as describedwith bleach and DEPC-water before eachuse. As ECs are polyploid and larger in sizeand visceral muscle cells are elongated, use70-µm filters and the largest nozzle size avail-able (100-µm) for the flow cytometer whensorting ECs or visceral muscle. Also, a longerdissociation time (1.5 hr) is needed for com-plete dissociation of ECs and visceral musclecells. The protocol has been verified under dif-ferent experimental conditions and works well

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for the cell types, with esg+ cell sorting be-ing the most efficient. Use of collagenase orelastase is optional, depending on availability.The authors have found elastase to be more ef-ficient. For flies expressing membrane-boundGFP (mGFP), collagenase was found to atten-uate the GFP intensity, and thus use of elastaseis recommended in such cases. The RNA iso-lation kit is used for microdissected samples,which require much lower buffer volumes; thecolumns thus will not take >300µl at one time.The yield is better if one column is used tobind all the RNA instead of multiple columnsbefore proceeding to the washing and elutionstep. To obtain maximum yield, keep all con-ditions RNase-free at all times and thoroughlyclean the sorter with bleach and alcohol be-fore starting the procedure. Clean sorter withDEPC-water in between samples of differentgenotypes. The RNA amplification procedureinvolves a two-step amplification of RNA andmight be costly if experiments are done inreplicates. The amplification step should bebypassed in cases where enough RNA is ob-tained after RNA isolation (e.g., for esg+ cellsor ECs) for downstream analysis. Amplifyequal amounts of RNA and amplify the repli-cates of one genotype in different batches toavoid amplification biases due to handling.

Anticipated ResultsThis protocol generates �200 to 300 ng of

total RNA for the esg+ cells, 300 to 400 ng forenterocytes, and <50 ng for the rest of the celltypes namely intestinal stem cells (ISCs), en-teroendocrine cells (EEs), enteroblasts (EBs),and the visceral muscle (VM). When 2 ngRNA is amplified, yields ranging from 50 to125 µg are obtained, which is sufficient formicroarray, RT-PCR, and RNA sequencing.

Time ConsiderationsThe dissection of midguts should be accom-

plished within 2 hr followed by cell dissoci-ation by enzymatic treatment for 1 to 1.5 hrdepending on cell type. The gates for FACSsorting should be pre-set to avoid any delayin the procedure. Dissociated cells are FACSsorted and RNA should be isolated from thesorted cells immediately, which takes 4 to 5 hr.All procedures from dissection up to RNAisolation (see Basic Protocol 1) take in total�10 hr and should be performed on the sameday to obtain high yields of RNA. The timelineof this part of the protocol should be carefullyplanned beforehand. The isolated RNA can bestored at –80°C until amplification. Amplifi-cation of RNA by standard methods takes 3

days (two 6-hr incubations for in-vitro tran-scription), including quantification by a Qubitfluorometer. The immunofluorescence stain-ing protocol starting from dissection to stain-ing takes 8 hr. Amplified RNA can be stored at−80°C until further gene expression profilingexperiments are performed.

AcknowledgementsThe authors thank Monika Laglotz from

the ZMBH FACS facility for help with thecell sorting, and David Ibberson at the CellNetworks-Deep Sequencing Core Facility ofUniversitat Heidelberg for help with RNAsequencing. The authors also thank HannaReuter for helping with the FACS dissociationprotocol, Jerome Korzelius for the Rab3-YFPpicture and scientific discussions, and ParthivePatel and Norman Zielke for generous helpwith flies and helpful insights. Funded by DFGSFB873, DKFZ A220, and ERC AdvancedGrant 26515 to B.A.E.

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