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s u p p l e m e n ta ry i n f o r m at i o n
www.nature.com/naturecellbiology 1
DOI: 10.1038/ncb1872
Figure S1 Immunofluorescence with 17.8 anti-Mili monoclonal antibody of mouse testis. Mouse testis sections stained with 17.8 anti-Mili monoclonal
antibody (green) and counterstained with DAPI (blue). Arrow shows a chromatoid body; scale bar = 10µm
Kirino et al, Supplement, page 6 of 16
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Figure S2 piRNPs do not associate with spliceosomes or the hnRNP or SMN complexes. Immunoprecipitations were performed from mouse testis with the following antibodies: Nonimmune mouse serum “NMS” (negative control); Y12 antibody “Y12”; anti-trimethylguanosine “TMG”, which recognizes the trimethylated small nuclear RNAs (snRNAs) and immunoprecipitates spliceosomal snRNPs; “4F4”, an anti-hnRNP C antibody that immunoprcipitate hnRNP complexes; and anti-SMN complex antibodies: “2B1”, anti-SMN; “12H12” anti-Gemin3; “10G11”, anti-Gemin5. RNA was isolated from the immunoprecipitates or from mouse testis total RNA,
dephosphorylated with calf intestinal phosphatase to remove 5'-phosphates and was then either 5’-end labeled with [γ-32P] ATP and T4 PNK or 3’-end labeled by [32P] pCp and T4 RNA ligase. Labeled RNA was resolved by electrophoresis on 15% denaturing polyacrylamide gels. Lane marked “M” contains 32P-labeled pBR322/MspI digest as size marker; nucleotide sizes are indicated on the left. piRNAs are not visible in the 3'-end-labeled Y12 lane in this exposure because the 3'-termini of piRNAs are 2'-O-methylated and they are poor substrates for T4 RNA ligase 9. 3’-end labeled snRNAs are present in the Y12 and TMG immunoprecipitates.
Kirino et al, Supplement, page 7 of 16
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Figure S3 Y12 immunoprecipitates piRNAs but not miRNAs. Northern blots with indicated probes of total RNA from mouse testis or RNA from indicated immunoprecipitates
Kirino et al, Supplement, page 8 of 16
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Figure S4 Dendrogram of mouse, Xenopus and Drosophila Piwi family proteins. Accession numbers of protein and translated ESTs used to construct the phylogenetic tree: Miwi: BAA93705; Mili: BAA93706; Miwi2: NP_808573;
DmPiwi: AAD08705; DmAub: CAA64320 and DmAgo3: ABO26294. IDs of EST sequences from Xenopus tropicalis Gurdon EST database are Xiwi: Xt6.1-CAAO5145.3.5; Xili: Xt6.1-CAAN4954.5.5 and Xiwi2: Xt6.1-CAAM16059.3.5.
Kirino et al, Supplement, page 8 of 16
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Figure S5 mRNA levels of Piwi, Aub and Ago3 are not altered in csul ovaries. mRNAs of Drosophila Piwi, Aub and Ago3 proteins from wild-type, heterozygous “+/-” or homozygous “-/-” csul ovaries were analyzed by qRT-PCR. The average ratios relative to wild-type from five independent experiments are shown; n=3 and s.d. shown
Kirino et al, Supplement, page 9 of 16
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Figure S6 Full scans for indicated figures
Kirino et al, Supplement, page 10 of 16
Supplementary Figure 6
Full scans for indicated figures
© 2009 Macmillan Publishers Limited. All rights reserved.
Supplementary Tables Supplementary Table 1 Peptide sequences obtained from mass spectrometry of proteins from immunoprecipitations of mouse testis Y12 immunoprecipitation Protein Amino acid number Peptide
Miwi 54-62 GMVVGATSK
744-748 RVNAR
749-756 FFAQSGGR
Mili 116-130 SSLPDPSVLAAGDSK
626-633 IAGPIGMR
17.8 (anti-Mili) immunoprecipitation Protein Amino acid number Peptide
Mili 51-64 KPEDSSPPLQPVQK
116-130 SSLPDPSVLAAGDSK
131-140 LAEASVGWSR
219-230 GTPQSLGLNLIK
283-290 LQQVVELK
294-303 KTDDAEISIK
537-549 ISQNETASNELTR
618-625 ELVNMLEK
626-633 IAGPIGMR
653-663 TIQSLLGVEGK
822-833 TVANYEIPQLQK
© 2009 Macmillan Publishers Limited. All rights reserved.
Supplementary Table 2 Putative sDMA motifs (GRG, ARG/GRA) present within animal Piwi proteins and plant Ago proteins; numbers refer to amino acid positions.
Species Proteins Number of potential sDMAs
sDMA motifs
Hiwi 10 3-GRARARARGRARG, 48-GRGRQRG, 728-GRG
Hili 6 40-GRG, 46-GRG, 96-GRG, 101-GRG, 145-GRG, 157-GRA
Hiwi3 7 3-GRARTRARGRAR, 63-ARG H.sapiens
Hiwi4 3 3-GRAR, 9-ARG Miwi 10 3-GRARARARGRARG, 48-GRGRQRG, 729-GRG
Mili 7 38-GRA, 44-GRG, 94-GRG, 99-GRG, 143-GRG, 155-GRA, 162-GRG M.musculus
Miwi2 4 66-GRARVRARG Xiwi 9 3-GRARARARGRARG, 55-GRGRQRG Xili 5 12-GRG, 19-GRG, 63-GRAR, 82-GRG X.tropicalis Xiwi2 8 3-GRARARARGRARG, 45-GRGR Ziwi 8 3-GRARARSRGRGRG, 46-GRGR
D.rerio Zili 13 36-GRARG, 51-GRGRA, 67-GRG, 83-ARG, 96-GRGRG,
113-GRG, 174-GRG, 220-GRG, 227-GRA, 237-GRG Piwi 2 6-GRGR Aub 4 10-ARGRGRGR D.melanogaster Ago3 4 3-GRG, 67-GRGRAR
C.elegans Prg1 4 6-GRGRGRG, 416-ARG Schmidtea
mediterranea (Planaria)
Smedwi-3 6 8-GRGR, 99-GRGRG, 134-GRGRG
Ago1 9 29-GRG, 47-GRG, 58-GRGGRG, 82-GRGRG, 93-GRG, 100-GRG, 1014-ARG
Ago2 9 9-GRG, 13-GRGRGGRG, 42-GRGRG, 57-GRG, 78-GRG, 111-GRG
Ago3 23
9-GRG, 13-GRGRG, 23-GRG, 28-GRG, 32-GRGRG, 45-GRG, 51-GRG, 59-GRG, 64-GRG, 68-GRGRG, 78-GRG, 83-GRG, 89-GRG, 97-GRG, 102-GRGRG, 128-GRG, 232-GRG, 252-GRG, 1051-GRA
A.thaliana
Ago5 9 15-GRG, 44-GRG, 52-GRGRG, 59-GRG, 81-GRGRG, 152-GRG, 319-GRG
© 2009 Macmillan Publishers Limited. All rights reserved.
Supplementary Table 3 Peptides sequences obtained from mass spectrometry of proteins from Y12 immunoprecipitations of Xenopus laevis oocytes Protein Amino acid number Peptide
Xiwi 304-311 DFADAVTK
345-357 SDGSDISFVDYYR
406-419 NDFGVMRDLAVHTR
526-533 VAQQIGMR
598-611 TLSKPQTVLSVATK
651-661 SIAGFVASMNR
756-763 FFAHLGGR
Xili 49-59 VQQASDFSTER
751-758 MVVIVVQK
© 2009 Macmillan Publishers Limited. All rights reserved.
Kirino et al, Supplement, page 1 of 11
Supplementary Results
Xenopus laevis piRNA analysis
Lower bound estimates on the number of X. laevis piRNAs
Deep sequencing of the Y12-immunopurified piRNAs from X. laevis and analysis by the
Illumina-provided toolset resulted in two sets comprising 270,596 (testis) and 663,226 (oocyte)
respectively. Approximately 96.7% of the testis reads were sequenced 3 or fewer times whereas ~85.4%
were sequenced exactly once. Similar observations held for the oocyte reads: ~ 92.5% of them were
sequenced 3 or fewer times whereas ~78.4% were sequenced exactly once.
Our earlier experience with high-throughput sequencing data has shown that a considerable
fraction of the reads cannot be located exactly in the genome of origin. This could be due to modifications
of the RNAs that occur prior to their being sequenced, errors introduced by the sequencing process itself,
or both. Generally, we have found that many more of the reported reads can be located in the genome if
one permits “editing” (typically replacement of one nucleotide by another suffices) at a small fraction of
the reads’ positions: for reads of the length discussed here, 10% of the length represents a reasonable,
albeit perhaps conservative, estimate.
The ability to locate the sequenced reads on the studied genome, in the presence of errors, could
in principle permit us to estimate a lower bound on the number of reads that correspond to true piRNAs.
However, in the absence of a genome sequence for X. laevis, this is not possible. Instead, we opted for
clustering of the reported that relied on their nucleotide sequence. This clustering process proceeded as
follows:
the reads of the set under consideration (i.e. testis and oocyte, in turn) are sorted in order of
decreasing number of copies that were sequenced;
initially, all reads of the set at hand are labeled ‘unprocessed’
let R represent be the next ‘unprocessed’ read
form a new read-cluster and assign R to be the cluster’s “leader”
compare R with the next ‘unprocessed’ read r in the set under consideration:
o if r and R agree identically on at least X% of the positions of the shorter of the two reads,
then
label r as ‘processed’, and
assign r to the cluster led by R
otherwise, do nothing
continue until no more ‘unprocessed’ reads remain
© 2009 Macmillan Publishers Limited. All rights reserved.
Kirino et al, Supplement, page 2 of 11
Using this procedure, we clustered the testis and oocyte reads for two choices of X: 90%, 80%. A
value of X=90% is in our estimate more representative of the combined impact that RNA editing and/or
sequencing error introduce to the reported sequences. The other choice, i.e. 80%, was meant to examine
how the number of resulting clusters will change if one increases the permitted tolerance.
At X=90% the 270,596 testis reads generated 198,200 clusters of one or more members,
suggesting that the sequenced reads were generally distinct from one another. This observation is
supported by the fact that the much more tolerant threshold of 80% results in only a small decrease in the
number (165,383) of formed clusters. Repeating the above analysis for the 663,226 oocyte reads gives
rise to 370,167 clusters of one or more members at X=90% and 279,074 clusters at X=80%.
The four sets of clusters (testis at 80% and 90%, oocyte at 80% and 90%) as well as the two sets
of reads we obtained from the Illumina analyzer are available for download at
http://cbcsrv.watson.ibm.com/piwi_modification/. In this directory, one can find the following files:
xl_testis.txt.gz
xl_oocyte.txt.gz
All the files contain plain text and have been compressed using the “gzip” utility. The files testis.txt and
oocyte.txt contain the sequenced reads sorted according to their copy numbers. The four files containing
the read clusters conform to the following format: …
…
>> CLUSTER 92
**532 TCAGAGAAAAAGCAGGGGACCATGGAAC
458 TCAGAGAAAAAGCAGGGGACCATGGAAT
132 TCAGAGAAAAAGCAGGGGACCATGGATC
90 CAGAGAAAAAGCAGGGGACCATGGAACA
36 GTCAGAGAAAAAGCAGGGGACCATGGAA
27 TCAGAGAAAAAGCAGGGGACCATGGAAA
24 TCAGAGAAAAAGCAGGGGACCATGGTCG
21 CAGAGAAAAAGCAGGGGACCATGGAATC
…
…
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Kirino et al, Supplement, page 3 of 11
The read that is marked with the “**” is the read leading the cluster, in this example
TCAGAGAAAAAGCAGGGGACCATGGAAC. The number preceding each read is the number of copies that were
obtained through high-throughput sequencing. Finally, the reads following the read-leader are the
cluster’s members.
Estimating the overlap of testis and oocyte piRNAs from X. laevis
We next attempted to estimate the overlap of the two sets of reads, testis and oocyte. In view of
the fact that the sequences at hand contain “errors” and in the absence of a genome on which we could
deposit the reads, this is not a straightforward question to answer.
One straightforward step would be to compare exact strings from the testis set with exact strings
from the oocyte set: doing so would underestimate the overlap, so one must treat the resulting number as
indicative of the degree of set overlap, and not representative. Considering the constraints of this
particular set of reads, we also carried out the following computation: we compared the reads from one set
to those of the other set allowing mismatches of at most Y% of the reads positions, with Y=5%, 10% and
20%.
When we compare the testis reads with the oocyte reads we find that at:
Y=0% 25,616 of 270,596 testis reads have counterparts in 25,616 of 663,226 oocyte reads
Y=5% 41,854 of 270,596 testis reads have counterparts in 61,382 of 663,226 oocyte reads
Y=10% 52,111 of 270,596 testis reads have counterparts in 86,090 of 663,226 oocyte reads
Y=20% 70,504 of 270,596 testis reads have counterparts in 133,714 of 663,226 oocyte reads.
The immediate conclusion from these comparisons is that the two sets of reads are largely
distinct, even at the rather tolerant threshold of 80%.
Evidence for a piRNA amplification ("ping-pong") loop
Analysis of the X. laevis piRNA reads showed that both the testis and oocyte collections, satisfy a
piRNA amplification (“ping-pong”) model.
Starting from the 5’ end and examining positions 1 through 10 inclusive of all 264,143 testis
piRNA reads reveals that there are only 150,294 unique 10-mers that span them. Of these 10-mers, 84,600
are found spanning these positions both as ‘sense’ (42,300) and as ‘antisense’ (42,300), and account for
more than 50% of all sequenced testis piRNA reads.
When we repeat this analysis for the oocyte collection, we find analogous results. Positions 1
through 10 inclusive from the 5’ end of all 659,831 oocyte piRNA reads are accounted for by 258,053
unique 10-mers only. Of these 10-mers, 206,894 are found spanning these positions both as ‘sense’
© 2009 Macmillan Publishers Limited. All rights reserved.
Kirino et al, Supplement, page 4 of 11
(103,447) and as ‘antisense’ (103,447), and account for nearly 2/3rds of all sequenced oocyte piRNA
reads.
To summarize, the majority of the piRNA reads in each of the two collections support the “ping-
pong” model of piRNA amplification in X. laevis. In order to facilitate further analyses by colleagues, we
have sub-selected and grouped those piRNA reads that support the model. In particular, we have created
two files, xl_ping_pong.testis.10.txt.gz and xl_ping_pong.oocyte.10.txt.gz that are available for
download at http://cbcsrv.watson.ibm.com/piwi_modification/. These two files contain plain text and
have been compressed using the “gzip” utility. Both files use the same format convention. File
xl_ping_pong.testis.10.txt.gz contains 42,300 groups of reads with each group being headed by a 10-mer
that is followed by two columns of piRNA reads: on the left column, we list those testis piRNA reads
where the 10-mer spans positions 1 through 10 as ‘sense’ whereas on the right column we list those reads
whose positions 1 through 10 contain the 10-mer as ‘antisense.’ An example such grouping is shown
next: the 10-mer and its reverse complement are shown underlined and in boldface. File
xl_ping_pong.oocyte.10.txt.gz contains analogous groupings but for oocyte piRNA reads (103,447
groupings in total). We expect that these two files will allow a practitioner to quickly identify relevant
groups of reads for his/her sequence of interest.
Example Grouping:
--------------------------------------------------------------------------
ACCAGACAAA
ACCAGACAAAGACCAAATGCTACAATCC TTTGTCTGGTTTTTGTAAAGGTGCAATA
ACCAGACAAAGAAATATACAGATATATA TTTGTCTGGTGAAGTGTTGCGATGGCTC
TTTGTCTGGTGAAGTGTTGCAATGGTCG
TTTGTCTGGTGAAGTGTTGCAATGGCTT
TTTGTCTGGTGAAGTGTTGCGATGGCTT
TTTGTCTGGTGAAGTGTTGCAATGGGTT
--------------------------------------------------------------------------
Reactivity of antibodies to sDMAs Although both Y12 and SYM11 antibodies recognize sDMAs, Y12 appears to have higher
affinity for Mili while SYM11 appears to have higher affinity for Miwi. This is likely due to the fact that
the epitopes that Y12 and SYM11 recognize differ in the configuration and spacing of GRG, ARG and
GRA triplets 1 2 3.
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Kirino et al, Supplement, page 5 of 11
The weaker reactivity of SYM11 towards Piwi and Ago3 likely reflects the higher affinity of
SYM11 towards sDMAs that are found in tandem 2 3 and the levels of different Piwi proteins. Aub, which
reacts strongly with SYM11 contains four putative sDMAs in tandem whereas Piwi and Ago3 contain
two and three, respectively (Supplementary Table 2); and Ago3 is expressed in lower levels in
Drosophila ovaries than Aub or Piwi 4.
Supplementary Discussion
Piwi protein is essential for self-renewal and maintenance of germline stem cells in ovaries and
absence of the piwi gene leads to severe defects in oogenesis 5. Piwi is predominantly a nuclear protein
and associates with piRNAs that are antisense to transposons 4 6. In homozygous csul ovaries the Piwi
protein does not contain sDMAs and although Piwi protein levels are moderately reduced, oogenesis
proceeds normally, indicating that sDMA modification in Piwi proteins apparently is not required for
oogenesis. In contrast, loss of sDMAs of Ago3 and Aub in csul ovaries leads to severe reduction of the
Ago3 and Aub protein levels. Interestingly, both Ago3 and Aub are cytoplasmic proteins. Ago3 associates
with piRNAs that are in sense orientation to transposons, while Aub associates with piRNAs that are
antisense to transposons 4 6. The antisense piRNAs of Aub and Piwi target transposons for
endonucleolytic cleavage resulting in transposon degradation and the generation of antisense piRNAs
from the targeted transposons that are loaded to Ago3 protein 4 6. Ago3-bound sense piRNAs in turn may
target antisense strands of transposon-containing transcripts spawning new antisense piRNAs that are
loaded in Aub and Piwi proteins, leading to a piRNA generating cycle known as the ping-pong
amplification loop 6 4. How primary piRNAs are generated and the precise biogenesis of piRNAs remains
unknown. It is possible that sDMA modifications of Aub, Ago3 and Piwi proteins are required for
efficient biogenesis or amplification of piRNAs and the reduced amount of Aub, Ago3 and Piwi reflect a
reduction in piRNA production. Alternatively, sDMAs may be required to stabilize Piwi proteins. The
net effect of lack of sDMA modifications of Piwi proteins is reduction of the levels of piRNAs, especially
the ones that associate with Aub and Ago3 since Aub and Ago3 protein levels are drastically reduced, and
upregulation of transposon levels. Maternally inherited Aub is required for specification of germ cells in
the developing embryo 7 and maternally inherited piRNAs are important for suppressing transposons in
the germline of the developing embryo 8. A likely mechanism to account, at least in part, for the
grandchildless phenotype of csul mutants may thus be the reduction of maternally inherited Aub, and also
Ago3 and Piwi, and their bound piRNAs.
© 2009 Macmillan Publishers Limited. All rights reserved.
Kirino et al, Supplement, page 6 of 11
Supplementary Methods
Antibodies
To produce a monoclonal antibody against Mili, full-length GST-Mili protein was purified from
baculovirus infected Sf9 cells by using Glutathione Sepharose 4B resin (GE Healthcare–Amersham
Biosciences), and used as antigen to immunize mice. Injections, hybridoma production, screening, and
ascites production were done as described previously 10, resulting in the “17.8” anti-Mili antibody.
For western blotting and immunoprecipitation, the 17.8 anti-Mili, Y12, anti-Flag (Sigma), anti-
sDMA (SYM11; Millipore), anti-aDMA (ASYM24; Millipore), anti-TMG (Santa Cruz Biotech), anti-β-
tubulin (Developmental Studies Hybridoma Bank), anti-hnRNPC (4F4), anti-SMN (2B1), anti-Gemin3
(12H12) and anti-Gemin5 (10G11) were used. Monoclonal antibodies Y12, 2B1, 4F4, 2B1, 12H12 and
10G11 were gifts from G. Dreyfuss. Antibodies against the Drosophila Ago1, Aub, Piwi and Ago3 were
gifts from MC. Siomi and H. Siomi 4, 11, 12.
Western blots and immunoprecipitations
Cell lysates were prepared from mouse testis (Pel-Freez Biologicals), Xenopus laevis oocytes,
testis, liver or Drosophila ovaries in a lysis buffer (20 mM Tris-HCL pH 7.5, 200 mM NaCl, 2.5 mM
MgCl2, 0.5 % NP-40, 0.1 % Triton-X100 and complete EDTA-free protease inhibitors (Roche)). Western
blots were performed as previously described 13. 17.8 anti-Mili ascites was used at 1:500 dilution.
Immunoprecipitations were performed essentially as described 14 15 .For immunoprecipitation of Mili
proteins, 5 µl of 17.8 anti-Mili ascites was used with 20 µl of protein-G agarose (Invitrogen). For
immunoprecipitations of Flag-tagged proteins, anti-Flag M2 agarose (Sigma) was used.
RNA isolation, labeling and β-elimination
RNA isolation, labeling and β-elimination were performed as previously described9. Briefly,
RNA was isolated from tissues or immunoprecipitates using Trizol (Invitrogen) and treated with calf
intestinal phosphatase (CIP; New England Biolabs). The 5’-dephosphorylated RNAs were then subjected
either to 5’- end labeling using [γ-32P] ATP and T4 polynucleotide kinase (T4 PNK, New England
Biolabs), or 3’- end labeling using [32P]pCp (GE Healthcare) and T4 RNA Ligase (New England
Biolabs). The labeled RNAs were resolved by 15 % PAGE containing 7 M urea and were visualized by
storage phosphor autoradiography using a Storm 860 PhosphorImager and ImageQuant software (GE
Healthcare). Sodium periodate (NaIO4) reaction was performed by incubating RNA in 50 µl of 10 mM
NaIO4 at 0 °C for 40 min in the dark. Five µl of 1 M rhamnose was added to quench unreacted NaIO4 and
incubated at 0 °C for additional 30 min. β-elimination was then performed by adding 55 µl of 2 M Lys-
© 2009 Macmillan Publishers Limited. All rights reserved.
Kirino et al, Supplement, page 7 of 11
HCl (pH 8.5) followed by incubation at 45 °C for 90 min. The treated RNAs were collected by ethanol
precipitation and resolved by 15 % PAGE containing 7M Urea. A 5’-labeled 28 nt synthetic RNA with
mouse piR-3 sequence (5’ 32P-UGAGAGUGGCAUCUAAAUGUUUAGUGGU-OH 3’) was used for a
control.
Northern blot analysis
Total RNA or RNA extracted from immunoprecipitates was resolved by 15 % PAGE containing
7 M urea, transferred to Hybond N+ membrane (GE Healthcare) and hybridized to 5’-end labeled probes,
antisense to mouse piR-1 (5’-AAAGCTATCTGAGCACCTGTGTTCATGTCA-3’) and miR-16 (5’-
CGCCAATATTTACGTGCTGCTA-3’), and Drosophila #1 roo antisense rasiRNA (5’-
TGGGCTCCGTTCATATCTTATG-3’), miR-8 (5’-GACATCTTTACCTGACAGTATTA-3’) and 2S
RNA (5’-TACAACCCTCAACCATATGTAGTCCAAGCA-3’) as described in 16.
Recombinant proteins and cDNA constructs
Recombinant Flag-Miwi and Flag-Mili (Figure 1e) and GST-Mili (used as immunogen to
generate anti-Mili monoclonal 17.8) were produced in baculovirus infected Sf9 cells. Briefly, for Flag-
Miwi, nucleotides 159 to 2744 of AB032604 were subcloned into the EcoRI/XbaI restriction sites of
pFASTBAC-FLAG(tev). For Flag-Mili, nucleotides 198 to 3113 of AB032605 were subcloned into the
EcoRI/XbaI restriction sites of pFASTBAC-FLAG(tev); all constructs were verified by sequencing.
Baculovirus transfer vectors were transformed into DH10Bac cells and recovered bacmid DNA was
screened by PCR with M13 sequencing primers for proper transposition of the transfer vector into the
baculovirus genome. Positive bacmid DNAs were transfected into Sf9 cells and passage 1 (P1) virus
stocks were recovered 96 hours post-transfection. A high-titer P2 virus stock was generated by infecting
Sf9 at an MOI of ~0.1, followed by incubation for 96-120 hours. For productions, 1 x 106 Sf9 cells/ml of
Sf900-II medium (Invitrogen) were infected with viruses for the Flag-Miwi and Flag-Mili proteins,
respectively, at an MOI of 1. Infected cells were harvested 48 hours post-infection and the recombinant
proteins were purified by immunoprecipitation using anti-Flag M2 agarose (Sigma).
The cDNAs for Flag-Miwi and Flag-Mili and Flag-Miwi truncations mutants (BC for 68-862aa of
Miwi and NP for 1-212aa) were gifts from S. Kuramochi-Miyagawa and T. Nakano 17. Full-length or
deletion mutants of Flag-Miwi (Figure 1f) were produced in 293T cells by vector transfection using
Lipofectamine 2000 (invitrogen).
Drosophila stocks
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Kirino et al, Supplement, page 8 of 11
csul flies (csulRM: w–;csulRM50/CyO), were a gift from J. Anne 18 and a deletion that uncovers csul
(wa Nfa-g; Df(2R)Jp7, w–/CyO) was obtained from Bloomington Drosophila Stock Center (Indiana
University).
Xenopus laevis
Oocytes were isolated from ovaries and defolliculated as described in 19. Testis and liver tissues
were procured from euthanized animals.
piRNA sequencing
Y12-immunopurified X. laevis piRNAs from testis and oocytes were 5'-end labeled and gel
purified. Directional ligation of adapters and cDNA generation was performed using the small RNA
sample prep kit (Illumina). Deep sequencing was performed on an Illumina Genome Analyzer.
Quantitative RT-PCR analysis
Total RNA sample from Drosophila ovaries was first treated with RQ1 DNase (Promega). 0.2 µg
of DNase-treated total RNA was used to reverse transcribe target sequences using each gene-specific
reverse primer (described below) and SuperScript II reverse transcriptase (Invitrogen). The resulting
cDNA was analyzed by quantitative RT-PCR performed by LightCycler 480 instrument (Roche) using
LightCycler 480 SYBR Green I master (Roche). Relative steady-state mRNA levels were determined
from the threshold cycle for amplification using the 2-CT method 20. RP49 was used as a control. The
following primer pairs were used for the RT-PCR; Piwi, forward (5’-
TGGACAGCAGAACATCGTGTTTC-3’) and reverse (5’-AGTAGAGTTCGGAGTTCATGG-3’); Aub,
forward (5’-AGCGTGCAGTAATGGGTATGGT-3’) and reverse (5’-
CGCGAATGATTATGTTGTATCGC-3’). Ago3, forward (5’-CATGAAATCGACAGTGGCTTGGA-3’)
and reverse (5’-ATGTGTCTATAAGCCTAGCACGTC-3’); HeT-A, forward (5’-
CGCGCGGAACCCATCTTCAGA-3’) and reverse (5’-CGCCGCAGTCGTTTGGTGAGT-3’); Rp49,
forward (5’-CCGCTTCAAGGGACAGTATCTG-3’) and reverse (5’-ATCTCGCCGCAGTAAACGC-
3’).
Immunofluorescence and confocal microscopy
Drosophila ovaries were dissected from adult flies in Robb's buffer. Immunostainings were
performed using monoclonal antibodies against Aub (purified, at 1:500 dilution), Piwi (supernatant, neat)
and Ago3 (purified, at 1:250 dilution) 4, as primary antibodies, and Alexa 594-conjugated anti-mouse IgG
(Molecular Probe) as the secondary antibody. Briefly, ovarioles were disaggregated from the dissected
© 2009 Macmillan Publishers Limited. All rights reserved.
Kirino et al, Supplement, page 9 of 11
ovaries and fixed for 20 minutes in 4% Formaldehyde/PBS, washed extensively with PBST-5 (PBS +
0.5% Triton X-100), permeabilized and blocked for 2 hours in PBST-5 containing 5% goat serum and
incubated with primary antibody overnight in the cold room at appropriate dilution in PBST-5-B (PBS +
0.5% Triton X-100 + 0.2% Tween-20 + 1% BSA). After extensive washes with PBST-5, ovarioles were
incubated with secondary antibody diluted in PBST-5-B for 2 hours, followed by extensive washes with
PBST-5 and PBS containing 0.2% Tween-20 and mounted. All images were acquired with a Zeiss LSM
510META NLO confocal microscope and identical magnification and settings were used for the pictures
shown in the same panel. For Figure 4e the following specifications, settings and magnification were
used: Wave length : Red = 543nm (1mW power, 100% transmission), blue (Dapi)=740nm (1W power,
2.5% transmission). Objectives: Plan Apochromat 20X/0.8(NA, numerical Aperture). Scan Zoom :
1.0Stack size : X = 450µm, Y = 450µm. Pixel : 1024 X 1024. Pinhole : 49µm for Red, 1000µm for Dapi.
Amplifier Gain : Red = 1.0, Dapi = 1.0. Amplifier Offset: Red = -0.06, Dapi = -0.07 (cutoff background,
like a threshold). Detector Gain : Red = 751, Dapi = 786.
For immunofluorescence of mouse testis with 17.8 anti-Mili antibody, testis from 3-month-old
B6 mice were fixed in 4% paraformaldehyde for 18 hours. The tissue was embedded in paraffin, 5µm
sections were cut, deparaffinized and treated with 10 mM sodium citrate buffer pH 6.0 at 950 C for 10
minutes for antigen retrieval. 17.8 (anti-mili) ascites was used at 1:100 dilution with anti-mouse Alexa-
488 Fab fragments (Invitrogen) for detection; nuclei were stained with DAPI. Images were acquired with
a Zeiss LSM10-META Confocal Laser scanning microscope.
In situ hybridization
The steps for colorimetric in situ hybridization were as described in 21 but with the following
variations. A Locked Nucleic Acid (LNA)-modified probe was designed against XL-piR-3, a frequently
sequenced X. laevis oocyte piRNA (XL-piR-3: 5'- UAAGUAGAAGAGCACCAAUGUCAUGUCC). The
sequence of the LNA probe was: 5'- ggAcatgaCattggtgcTcttctActta -3'-DIG (capital letters: LNA-
modified nucleotides; DIG: digoxigenin) and was synthesized by IDT. Defolliculated oocytes were fixed
with 10% Neutral buffered formalin, paraffin embedded and sectioned at 5µm. Slides were deparaffinized
in three changes of fresh xylene, rehydrated in graded alcohols 10 min each (100%, 100%, 95%, 95%,
70% and 50%), followed by two washings for 5 min distilled water (ddH2O) and then treated with 10 mM
sodium citrate buffer pH 6.0 at 950 C for 10 minutes, in lieu of proteinase K digestion. The slides were
cooled for 30 min at room temperature (RT) and washed three times for 5 min each with ddH2O, once
with PBS and prehybridized with 4x SSC, 3% BSA buffer at 480 C. The hybridization buffer (4x SSC,
10% dextran sulfate) was pre-warmed at 480 C. Hybridization was performed in hybridization buffer
© 2009 Macmillan Publishers Limited. All rights reserved.
Kirino et al, Supplement, page 10 of 11
containing 5 pmol of probe at 480 C for 5 hours. Slides were washed in a solution containing 4x SSC, 0.1
% Tween-20 at 52oC for and then for 5 min with agitation in 2x SSC buffer at 52oC, 5 min in 1X SSC
buffer at at 52oC and another 5 min in PBS at RT. Slides were incubated at RT in 100 mM Tris pH 7.5,
150 mM NaCl, 1% blocking reagent (Roche), 0.5% Triton and 1 mM Levamisole (to block endogenous
alkaline phosphatase) for 1 h. Fresh blocking buffer containing a 1/1000 dilution of anti-digoxigenin-
alkaline phosphatase Fab fragments (Roche) was applied overnight at 40 C. Slides were washed 4 times
for 10 min each in PBS/0.1 % Tween-20 (PBST) and washed 2 times for 10 min each in Staining solution
containing 10 mM Tris-HCl pH 9.0, 50 mM MgCl2, 100 mM NaCl, 0.1 % Tween-20, followed by
NBT/BCIP developing solution (10 ml Staining solution, 48 µl of 50 mg/ml NBT, 35 µl of 50 mg/ml
BCIP). Incubation in developing solution was usually complete by 2 hrs. Slides were rinsed in PBS,
ddH2O and were dehydrated by passing through a series of alcohols (50%, 75%, 95%, 100%, 100%) and
xylenes and coverslipped in PermaMount. Images were obtained using Leica LM LB2 microscope with a
digital camera Leica DFC-480.
© 2009 Macmillan Publishers Limited. All rights reserved.
Kirino et al, Supplement, page 11 of 11
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© 2009 Macmillan Publishers Limited. All rights reserved.