SiRNA transfection, northern blotting and RT-qPCR analysis were performed as

Transcription

1 Supplemental Material: Supplemental Materials and Methods Supplemental Figures S1 to S7 Supplemental Tables S1 and S Additional References Supplemental Materials and Methods Knockdown, northern blotting and RT-qPCR analysis SiRNA transfection, northern blotting and RT-qPCR analysis were performed as previously described (Saito et al. 1). SiRNAs and DNA oligonucleotides used are summarized in Supplemental Table S. Production of the anti-dmgtsf1 antibody DmGTSF1 fused to GST was used as the antigen to immunize mice. An anti-dmgtsf1 monoclonal antibody was produced essentially as previously described (Saito et al. ). Western blotting was performed as previously described (Saito et al. ). Anti-tubulin was obtained from the Developmental Studies Hybridoma Bank and was used at 1:1, dilution. Immunofluorescence Immunostaining of ovaries and OSCs was performed as previously described (Saito et al. ; Saito et al. 9). The primary antibodies used were: culture supernatants of anti-piwi hybridoma cells (PD; 1:1 dilution) (Saito et al. ), a purified mouse 1

2 monoclonal antibody of anti-aub (1:5 dilution) (Nishida et al. 7), a mouse monoclonal antibody for the myc-tag (1:1, dilution; Sigma), a rabbit polyclonal antibody for the Myc-tag (1:1, dilution; Sigma), a mouse antibody to FasIII (1:5 dilution), and a mouse antibody to Orb (1: dilution; the Developmental Studies Hybridoma Bank). The secondary antibodies used were: Alexa -conjugated anti-mouse IgG (Molecular Probes), Alexa 5-conjugated anti-mouse IgG (Invitrogen), Cy3-conjugated anti-mouse IgG, Alexa -conjugated anti-rabbit IgG (Molecular Probes), Alexa 5-conjugated anti-rabbit IgG (Invitrogen), and Cy3-conjugated anti-rabbit IgG. DNA was stained with,-diamidino--phenylindole (DAPI). All images were collected using a confocal microscope (Zeiss LSM71). Plasmid construction and rescue assays in OSCs To obtain a cdna encoding DmGTSF1, poly(a)+ RNAs were purified from the ovaries of flies of the yellow-white Drosophila strain, and RT-PCR was carried out using DmGTSF1-F and DmGTSF1-R (primers are shown in Supplemental Table S). The entire sequence of DmGTSF1 was cloned into pbluescript II (SK+) vector and analyzed. To produce DmGTSF1 expression vectors, full-length DmGTSF1 cdna was amplified by using DmGTSF1-F-EcoRI and DmGTSF1-R-XhoI, and then inserted into a pgex-5x1 expression vector (GE Healthcare Bioscience) and pacm vector (Saito et al. ). To yield EGFP-fused DmGTSF1r, PCR fragments were amplified with the DmGTSF1r-Fu-F and DmGTSF1r-Fu-R. The PCR fragments were incubated with the InFusion reaction enzyme and pacm-egfp vector (Saito et al. 1) digested with Hind III and Eco RI. To yield -resistant DmGTSF1 (DmGTSF1r) and cysteine to alanine mutants, PCR-based mutagenesis was employed. The primers and PCR

3 enzymes used are listed in Supplemental Table S. The rescue assays were performed as previously described (Nishimasu et al. 1). RNA sequencing and data analysis Poly(A)+ RNA libraries were prepared for sequencing using standard Illumina protocols (using the multiplex library preparation method), and analyzed using two lanes of Illumina HiSeq. This yielded ~1 3 million genome-mapped reads in each transfected sample. For computational analyses, we first trimmed for adaptor sequences using Cutadapt, extracted high quality bases from each read ( 11 nt), and mapped these to the Drosophila genome release dm3 from UCSC using Bowtie (v..) (Langmead and Salzberg 1) with default parameters. For calculation of reads per kilobase per million mapped reads (RPKM) of transposable elements, Drosophila genome (dm3) and transposable element consensus (obtained from Repbase (Jurka 199)) mapped results were processed using an in-house Perl script. The numbers of reads falling within transposable elements were counted and normalized to the count of genome-mapped reads and the length of each transposable element. RPKM values for transcripts were calculated using TopHat v..7, (Trapnell et al. 9) referring to the transcriptome annotation available in Illumina igenomes ( A list of genes with mapped TE insertions either in the gene body or in close proximity (up to 5 kb away), and upregulated in Piwi -knockdown OSCs, was obtained from a previous publication (Sienski et al. 1). All heatmaps were generated using Java Treeview (Saldanha ). For the heatmap shown in Figure 3B, the 75 transposable elements with the highest fold change in the Piwi- sample (versus levels in the control- 3

4 sample) are shown. Sequencing data is available through Gene Expression Omnibus (accession no. GSE7). Immunoprecipitation and western blotting DmGTSF1 expression vectors were transfected into OSCs using Xfect transfection reagent (Takara). The OSCs were lysed in lysis buffer [ mm Tris-HCl (ph.), 1 mm KCl, 5 mm MgCl, mm DTT,.1% Triton X-1, and protease inhibitors]. Anti-Myc antibodies (Sigma) were immobilized on GammaBind G sepharose (GE Healthcare). Reaction mixtures were rocked at C for 3 h and the beads were washed five times with lysis buffer. Total proteins were separated on SDS-PAGE gels and western blotting was performed as previously described (Saito et al. ). To check the efficiency of the immunoprecipitation analyses, we used the mouse IgG TrueBlot ULTRA (ebioscience) to discriminate the Myc-tagged proteins from the IgGs used in the immunoprecipitation assay. GST pull-down assay GST pull-down assays were performed as previously described (Saito et al. 7). Briefly, GST-DmGTSF1, GST-Pimet/DmHen1 or GST itself immobilized on glutathione sepharose B (GE Healthcare) was incubated with OSC lysate for three hours in a cold m and extensively washed with a buffer containing mm Tris-HCl (ph.), 1 mm KCl, 5 mm MgCl, mm DTT, and.1% Triton X-1. The eluates were then probed with antibodies against Piwi, Armi, and AGO1.

5 A Mouse GTSF1 (17aa) Mouse GTSF1-like (151aa) Drosophila CG393/DmGTSF1 (17aa) Drosophila CG35 (1aa) Drosophila CG33 (153aa) Drosophila CG13 (93aa) U11-K-like CHHC type Zn finger Ohtani et al Figure S1 Mouse GTSF1 --MEDTYIDSLDPEKLLQCPYDKNHQIRACRFPYHLIKCRKNHPDVANKLATCPFNARHQ Mouse GTSF1-like MEPESIEICPYNPHHRIPLSRFQYHLASCRKKNPKKAKKMASCKYNACHV DmGTSF MVYCPYNKEHKMLRKKLQQHILKCRVIYKD-TVELMVCPFNSSHL Dm CG MSADPKLYEYVNCPNSSSHLVMLFKMPYHLPLCAKKFP--SDELARCPYNRTHM Dm CG33 MSKSNASVEGPRFEEHLVCPFDGLHRILPLDMKTHVLQCAKNFP--LEKLVHCPYNCGQI Dm CG MSDFEYGICPYDKSHRILLFRMPKHLIKCEKNYC--GPPLQTCKYN--AT Mouse GTSF1 VPRAEISHHISSCDDKSCIEQDVVNQTRNLGQETLAESTWQCPPCDEDWDKDLWEQTSTP Mouse GTSF1-like VPIRKLAEHEATCVNR-----SSVEEEDTLGPLQVSLPQPQNQDTLQVRWLSNPDIWNVD DmGTSF1 IPEPQFFQHTQSCEDR-----NIIVHYQTSAPAVLSEDTRHAK----IESEENWDDDESV Dm CG35 YPIADIYEHIIKCP SYLREESHSDAKEDAKDCDAKE-----SDAKDWDADPPV Dm CG33 HSVAEMENHINDCP DRAYLERNNVPSDELPSLAQRPRGVEVESEEDWDAEPPS Dm CG13 HRVQDMEKHLKECD YYLRSIEN Mouse GTSF1 FVWGTASFCGNNSPANNIVMEHKSNLASGMRVPKSLPYVLPWKNNGNAQ Mouse GTSF1-like GANCHPMFVLKSFVPQKLVCESDIQESRGGDQCPEDPQTRTRKANF DmGTSF1 PDYDPQVYCSRANIVREPNGLFPAQRRAFIEQEKRRHFGEDYEEEKKPPKAKARADLRPT Dm CG35 STYDPNIHCEKNPIIRRLHGATRSSRRAFREKERKRLMDLNTFP Dm CG33 QTYNPSLHCERSMVIRTIHGATRSARREFRKREQKRLNDLNP Dm CG QAVQIALRSRIPPKQEDGHDVDTPF B C relative expression sipiwi sidmgtsf1 sicg13 sicg35 sipiwi sicg33 Mouse GTSF Mouse GTSF1-like DmGTSF1 PYKHRRPYSRRQ Dm CG Dm CG Dm CG sipiwi sidmgtsf1 sicg13 sicg35 sipiwi sicg33 - Idefix pir-1 - endo-sirna sl1 relative expression 1 Supplemental Fig. S1. DmGTSF1, a Drosophila homolog of mouse GTSF1, is required for TE silencing but not for pirna biogenesis in OSCs. (A) Schematic drawing of the Drosophila and mouse GTSF1 family proteins (Top). Multiple sequence alignment of the amino-acid residues from GTSF1 family proteins (Bottom). The conserved cytosine and histidine residues were shown by yellow stars (1st Zn-finger motif) and blue stars (nd Zn-finger motif). (B) DmGTSF1 and three DmGTSF1 paralogs (CG13, CG35 and CG33) were depleted in OSCs by. The knockdown efficiency was confirmed by RT-qPCR (data not shown) in which the primers were shown in Supplemental Table S. Northern blotting performed on total RNAs isolated from the cells h post-transfection. The levels of Idefix-piRNA were unchanged by DmGTSF1 family proteins, suggesting the dispensability of DmGTSF1 in pirna biogenesis in OSCs. (C) RT-qPCR showed that was derepressed by DmGTSF1 depletion, as it was by Piwi depletion. However, depletion of three DmGTSF1 paralogs (CG13, CG35 and CG33) unaffected level. 5

6 Ohtani et al Figure S A B DmGTSF1 CG3 EGFP DmGTSF1 P{w[+mC]=GSV}GS19 (kda) DmGTSF1 15 C - Tubulin relative expression I element DmGTSF1-/- DmGTSF1+/- ovary 97 DmGTSF1-/- I element 97 I element carcasses 97 I element DmGTSF1+/- Supplemental Fig. S. (A) Schematic drawing of the Drosophila genomic locus containing the DmGTSF1 gene (green) and a neighboring CG3 gene (magenta). Insertion of a P-element in the DmGTSF1 gene is shown. The primer binding sites used in RT-PCR analysis were shown with arrowheads. (B) Western blotting shows that an anti-dmgtsf1 antibody raised in our laboratory specifically recognizes the antigen in OSC. An anti-tubulin antibody was used as a loading control. (C) DmGTSF1+/- or DmGTSF1-/- adult flies were dissected into ovaries and carcasses, from which total RNAs were isolated for RT-qPCR. A subset of TEs was derepressed in DmGTSF1-/- ovaries but not in the carcasses.

7 - Piwi R =.9 fold change in Piwi- (log) R =.9 fold change in Piwi- (log) E fold change in Mael- (log) 97 I-element - Mael - HP1a - DmGTSF1 sidmgtsf1 sipiwi fold change in HP1a- (log) fold change in Armi- (log) 97 I-element Piwi - Armi 3 1 fold change in DmGTSF1- (log) EGFP 97 I-element relative expression 5 B DmGTSF1 C A Ohtani et al Figure S3 - Tubulin D R =.5 Myc-Piwi siarmi sidmgtsf1 Myc fold change in Piwi- (log) DAPI R =.9 DIC fold change in Piwi- (log) Myc DAPI Merged DIC simael Myc-DmGTSF1 sihp1a Supplemental Fig. S3. (A) The results of mrna-seq are recapitulated by RT-qPCR analysis. The steady-state levels of transcripts from TEs were measured relative to RP9. (B) Scatter plot showing fold change of TEs of knockdown for Piwi, Armi, Mael, DmGTSF1, or HP1a to -knockdown sample for EGFP. R values are indicated. (C) Western blotting shows that knockdown of DmGTSF1 or Piwi did not affect the protein expressions of factors in the primary pirna pathway. (D) Depletion of Armi caused Myc-Piwi (red) to be mislocalized to the cytoplasm as described previously (Saito et al. 1). In contrast, the nuclear localization of Myc-Piwi (red) was unaffected by DmGTSF1 depletion in OSCs. (E) The nuclear localization of Myc-Piwi (red) was unaffected by Mael or HP1a depletion in OSCs. 7

8 Ohtani et al Figure S A CG15 CG157 CG39 CG13 Ira elk CG135 Cda5 ex CG159 CG11 bab1 Shal Cypa9 CHKov1 Cpr7Fb neo CG1159 CG55 p13cas CG177 CG37 CG13375 CG3393 Oat CG37 Sp7 CG171 Piwi (Sienski et al) Piwi Armi DmGTSF1 HP1a Mael TE within 5kb Juan Stalker Doc 1 1 Transpac Stalker fold change (log) 3 C kbp bp 3 Marker TE Neighboring Gene CG15 bab1 CG177 p13cas B TE Insertion *PCR signals are confirmed for TE insertion by direct sequencing FWD primer TE Neighboring Gene REV primer 7~95 bp product without TE insertion Supplemental Fig. S. (A) As described within Fig. 3C. TE insertions either in the gene body or in close proximity (within 5kb) of each gene are indicated (Sienski et al. 1). The arrows indicate genes that are tested for their TE insertion. CG15 (highlighted with red arrow) was a gene highly up-regulated by Piwi knockdown, where genes with blue arrows are those with lower up-regulation level. (B) Schematic figure describing PCR amplification of TE inserted regions. PCR primers are designed as shown in arrows to check TE insertion. ~bp product should be amplified without TE insertion and >kbp product should be amplified with TE insertion. (C) Result of PCR amplification of the TE harboring genomic regions. Each PCR product has been sequenced directly to confirm that each band consists of TEs described within (A).

9 Ohtani et al Figure S5 A Lysate Myc-EGFP Myc-DmGTSF1-EGFP IP (anti-myc) Myc-EGFP + Myc-DmGTSF1-EGFP - Myc-DmGTSF1-EGFP + RNase - Piwi - Myc- DmGTSF1- EGFP - Myc-EGFP B Lysate CBB GST - GST pull-down GST + GST-DmGTSF1 - GST-DmGTSF1 + RNase - Piwi 5 3 (kda) C Lysate GST pull-down GST GST-DmGTSF1 - Piwi D Myc-DmGTSF1 DAPI Mael Myc-DmGTSF1 Mael DAPI Myc-DmGTSF1 Mael DIC - Mael Supplemental Fig. S5. (A) Myc-DmGTSF1-EGFP and endogenous Piwi coprecipitated even in the presence of RNase A. (B) DmGTSF1 associates with PIWI in the presence of RNase A. (C) GST-DmGTSF1 associates with Piwi but not with Mael. Bound fractions with GST-DmGTSF1 were probed with antibodies against Piwi and Mael (Sato et al. 11). (D) The endogenous Mael was partially localized in the nucleus as previously reported (Sienski et al. 1). 9

10 A Myc DAPI Merged DIC Ohtani et al Figure S Myc-DmGTSF1r-CA-EGFP Myc-DmGTSF1r-C37A-EGFP Myc-DmGTSF1r-CA/C37A-EGFP B Lysate IP (anti-myc) Lysate IP (anti-myc) Myc-EGFP Myc-DmGTSF1r-EGFP Myc-DmGTSF1r-CA-EGFP Myc-DmGTSF1r-CA-EGFP Myc-DmGTSF1r-C37A-EGFP Myc-DmGTSF1r-C57A-EGFP Myc-EGFP Myc-DmGTSF1r-EGFP Myc-DmGTSF1r-CA-EGFP Myc-DmGTSF1r-CA-EGFP Myc-DmGTSF1r-C37A-EGFP Myc-DmGTSF1r-C57A-EGFP - Piwi Myc-EGFP Myc-DmGTSF1r-CA/C37A-EGFP Myc-EGFP Myc-DmGTSF1r-CA/C37A-EGFP - Piwi - Myc-DmGTSF1r-EGFP and mutants - Myc-EGFP - Myc- DmGTSF1- CA/C37A- EGFP - Myc-EGFP Supplemental Fig. S. (A) DmGTSF1 Zn-finger mutants localized in the nucleus as the naïve DmGTSF1 did (Fig. 1C). (B) DmGTSF1 Zn-finger mutants associate with Piwi. -resistant Myc-tagged full-length DmGTSF1r was fused EGFP at the C-terminal end to generate Myc- DmGTSF1r-EGFP. Myc-DmGTSF1r-EGFP and Zn-finger mutants were transiently transfected into OSCs and immunopurified using an anti-myc antibody. Endogenous Piwi was probed with anti-piwi antibody (Saito et al. ). Secondary HRP-conjugated TrueBlot anti-mouse antibody was used for the detection of the immunoprecipitated proteins (bottom right). 1

11 A signal density (RPM) B Pol II ChIP-seq 5 Myc-DmGTSF1 sipiwi simael sidmgtsf1 siarmi sipiwi Myc signal density (RPM) Ohtani et al Figure S7 DAPI 1 DIC signal density (RPM) 1 Supplemental Fig. S7. (A) Density plots for normalized Pol II ChIP-seq reads over three transposons;,, and, are shown. Gray distributions correspond to Pol II ChIP-seq signal levels in control knockdown (EGFP-knockdown), and colored lines correspond to levels in knockdown of Piwi, Mael and DmGTSF1. (B) Depletion of Piwi and Armi did not affect the nuclear localization of Myc-DmGTSF1 (red). DAPI staining (blue) shows the locations of nuclei in the cells. 11

12 Supplemental Table S1 Ohtani et al. Supplemental Table S1. mrna-seq library read count Genome Mapping Transposable Element Mapping Sample Treated sirnas Index Total reads Uniquely mapping reads Multiple mapping reads Total mapping reads Uniquely mapping reads Multiple mapping reads Total mapping reads EGFP (control) CAGATC,13,7 3,79,1 3,393, 33,7,7,3,13 193,71,535,95 sirna treated OSCs Piwi ACTTGA,77, 31,9,39,5,317 3,79, 3,57,91 51,59 3,,577 Armi GATCAG 31,39,93 19,735,15,57,1,37,339 1,,3 1,7 1,99,11 DmGTSF1 GAGTGG,91,93 7,7,5 3,3,57 31,,93,,75 1,9,,57 HP1a TAGCTT,1,19 15,515,9 3,9,3 1,5,5,777,73 15,77,93,75 Mael AGTCAA 37,5,3 1,75,1 3,7,3 5,393,3,79,193 19,7,93,5 Total -,53,5 17,1,17 1,351,9 1,39,3 15,9,7 1,133,171 1,,93 ChIP-seq library read count Sample sirna target Index Total reads Genome Mapping Transposable Element Mapping Uniquely Multiple Total Uniquely Multiple Total mapping mapping mapping mapping mapping mapping reads reads reads reads reads reads sirna treated OSCs, IP with Pol II Ab. EGFP(control) CGTGAT,39,3 3,1,51 7,35,11, 35,31,1 37,15 Piwi ACATCG,93,3,77,3 9,93 3,,97 3,3,93 39,17 Mael CACTGT,,377,9,77 1,7 3,313, ,93,91 35,7 DmGTSF1 TCAAGT,53, 3,3, 39,,1,77,71,9 3,1 Total - 17,77,11 1,5,57,53,3 15,11,97 1,5,59 9,35 1,5,17 1

13 Supplemental Table S Ohtani et al. Supplemental Table S. Primers and sirnas Experiment Primer Name Primer sequence Vector construction DmGTSF1-F DmGTSF1-R DmGTSF1-F-EcoRI DmGTSF1-F-XhoI ATGGTTTATTGCCCGTACAAC CTACTGGCGCCTTGAGTATG ATGAATTCATGGTTTATTGCCCGTACAAC ATCTCGAGCTACTGGCGCCTTGAGTATG Experiment Vector Name Primer Name System Primer sequence pacm-dmgtsf1r pacm-dmgtsf1r- EGFP DmGTSF1-mt-F DmGTSF1-mt-R DmGTSF1r-Fu-F DmGTSF1r-Fu-R PrimeSTAR In-Fusion AACCGAACGGGTTGTTTCCCGCCC CACGGACAATATTGGCCCTGGAACAGTAG AGACCTGAACAAGCTTATGGTTTATTGCCCGTACAA TATATCTGCAGAATTCCTGGCGCCTTGAGTATGG Vector construction pacm-dmgtsf1r- CA-EGFP pacm-dmgtsf1r- CA-F CA-R CA-F Site-directed mutagenesis kit Site-directed GAACAAGCTTATGGTTTATGCCCCGTACAACAAGGAGCAC GTGCTCCTTGTTGTACGGGGCATAAACCATAAGCTTGTTC CAGCAGCACATCTTAAAAGCCCGTGTGATCTACAAAGAC CA-EGFP CA-R mutagenesis kit GTCTTTGTAGATCACACGGGCTTTTAAGATGTGCTGCTG pacm-dmgtsf1r- C37A-EGFP C37A-F C37A-R Site-directed mutagenesis kit CACGGTGGAACTTATGGTTGCTCCCTTCAATAGTTCCC GGGAACTATTGAAGGGAGCAACCATAAGTTCCACCGTG pacm-dmgtsf1r- C57A-F Site-directed CTTTCAACACACCCAATCGGCCGAGGATCGAAACATTATAG C57A-EGFP C57A-R mutagenesis kit CTATAATGTTTCGATCCTCGGCCGATTGGGTGTGTTGAAAG Experiment Gene Primer sequence (Forward, Reverse) RT-PCR DmGTSF1 CG3 CTACGACGAATCCGATATG, CTACTGGCGCCTTGAGTATG ATGGGCAAGGGCTTCAACAA, AAATGCTGTACATGGTGCAC Genomic PCR CG15 bab1 CG177 p13cas GGGCGAGTTACAATGGGGAT, AAGGTAGTGAGCTGGCATACA TCTTTGCCGTCGTCTTGAGT, TGCCTTAAATACACGCCGCA CAATCCTCGCATCCTTCCGA, AGCTTATTTCGCGCTCGACT AAGACATCGCGTTCCAGTGT, CTGAACTTTCAACCCGCTGC RT-qPCR ChIP-qPCR RP9 97 I-element CG15 CG157 CG39 bab1 p13cas CG177 CG393 CG13 CG35 CG33 CCGCTTCAAGGGACAGTATCTG, ATCTCGCCGCAGTAAACGC CTGGCAAAGGGATTTCATCA, TGCATTCCTAAGGCCAAATG TATGGGCTGAGGCGATAAAC, CAAGTGGCTCACTGCTGGTA GACCAAATAAAAATAATACGACTTC, AACTAATTGCTGGCTTGTTATG CGCGCGGAACCCATCTTCAGA, CGCCGCAGTCGTTTGGTGAGT AACAGAAACGCCAGCAACAGC, CGTTCCCATGTCCGTTGTGAT CGTCTGCAATGTACTGGCTCT, CGGCACTCCACTAACTTCTCC TATCGCATGGCAGATAGCCAAA, CGTGGAATTCGGAAGTGGTTTC CTTAAAGGTTGTGGCGGGTA, TGTAGACAGCATCCTATTTCTTGC ACACCTTCTGGTGCCCTTCT, AGTTCCCTGGCAGCCTTAAT GTTTGCCAAGTGGTTCTGGT, CTGCTGTTGCTGTTGCTGTT ACAAGGAGTTGAAGCGCAAA, GGTGAAGGAGTGCGATCAAT GTCCACTGAGCAGCCTGATT, TGTCATCAGCGAACAGTCCC TCTTAGGCAGCAACCAGCTC, TCGAGCTGGAAAAGCTCAGG ATGATCACCTCGCCGCTATG, TAGAGCATGGGTCGGCAATC TGATACCAGAGCCCCAGTTC, GTGTCTGGTGTCTTCGCTGA TACGGAATTTGCCCTTACGA, GGTGTGTGGCGTTGTACTTG CCTACCATTTGCCGCTTTGT, GTGGCTCTCCTCGCGTAAGT TACACTCGGTTGCCGAAATG, TCCTCTTCGCTCTCCACCTC ATGCGAATTCAGGATGTACG, AGGGAGATCTCTTGTGACAGC GGCTCATTGCCGTTAAACAT, TCTTTCGCTGAGGTTCGTCT ACATGGGTGCATTGCTCAAA, CGGGCACATCTGCCTATCTT 13

14 Experiment Gene Probe sequence Northern Blot endo-sirna-sl1 tj-pirna Idefix-piRNA GGAGCGAACTTGTTGGAGTCAA GGTAATGGGAATGCACTTCTCTTGAA AAACTACTGGCAATCGTTTGGGAA Experiment sirna name sirna sense sirna antisense EGFP-si Piwi-si Armitage-si HP1a-si Maelstrom-si Spindle-E-si DmGTSF1-si CG13-si CG35-si CG33-si GGCAAGCUGACCCUGAAGUTT GCUCCCAGGCGUGAAGGUGTT CAGCUUAAGGCGAAUCCAATT GAUCUUGGGUGCCUCCGACTT CGCCAAGAUGUCCCAUGAUTT CUCGCGGCAUGUAAAGAUUTT CAUAGUGAGAGAGCCCAAUTT GCAUCUGAAGGAGUGUGACTT GGACUGUGAUGCCAAGGAATT CUGCGGUCAAAUACACUCGTT ACUUCAGGGUCAGCUUGCCTT CACCUUCACGCCUGGGAGCTT UUGGAUUCGCCUUAAGCUGTT GUCGGAGGCACCCAAGAUCTT AUCAUGGGACAUCUUGGCGTT AAUCUUUACAUGCCGCGAGTT AUUGGGCUCUCUCACUAUGTT GUCACACUCCUUCAGAUGCTT UUCCUUGGCAUCACAGUCCTT CGAGUGUAUUUGACCGCAGTT 1

15 Additional References Jurka J Repeats in genomic DNA: mining and meaning. Curr Opin Struct Biol : Langmead B, Salzberg SL. 1. Fast gapped-read alignment with Bowtie. Nature methods 9: Nishida KM, Saito K, Mori T, Kawamura Y, Nagami-Okada T, Inagaki S, Siomi H, Siomi MC. 7. Gene silencing mechanisms mediated by Aubergine pirna complexes in Drosophila male gonad. RNA 13: Saito K, Nishida KM, Mori T, Kawamura Y, Miyoshi K, Nagami T, Siomi H, Siomi MC.. Specific association of Piwi with rasirnas derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev : 1-. Saldanha AJ.. Java Treeview--extensible visualization of microarray data. Bioinformatics : 3-3. Trapnell C, Pachter L, Salzberg SL. 9. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 5: