SRX078290 qinHet SRP007101 qin small RNA sequencing Total RNA was isolated from manually dissected ovaries from 2-4 day flies using mirVana (Ambion, Austin, TX, USA). The RNA was quantified by absorbance at 260 nm. 18-30 nt RNAs was selected from 100 micrograms total RNA by running a 15% denaturing urea-polyacrylamide gel (National Diagnostics, Atlanta, GA, USA). 2S rRNA was depleted by hybridization using a complementary, immobilized DNA oligonucleotide (5'-biotin-TCA ATG TCG ATA CAA CCC TCA ACC ATA TGT AGT CCA AGC A-3'). Briefly, for each library 400 µl MyOne Streptavidin C1 Dynabeads (Invitrogen) were washed twice in 0.5× SSC (1× SSC: 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) at 4°C, then re-suspended in 400 µl 0.5× SSC. The beads were then loaded with 200 pmole DNA oligonucleotide at 4°C for 30 min, washed once in 0.5× SSC, re-suspended in 1 ml 0.5× SSC, and then warmed to 65°C for 5 min. Size-selected RNA (50 µg) was incubated at 80°C for 5 min, and then added to the pre-warmed beads and incubated at 50°C for 1 h. The beads were then removed by magnetic capture and the supernatant mixed with 3 volumes of ethanol, 0.3 M (f.c.) sodium acetate, pH 5.2, and 1 µl (20 µg/µl) glycogen (Roche, Indianapolis, IN, USA).100 pmole of 3' pre-adenylated adapter (5'-rAppTCG TAT GCC GTC TTC TGC TTG T/ddC/-3') was ligated to small RNAs using Mutant Rnl2_1-249_K227Q (17-23B) (Addgene, Cambridge, MA, USA) at 4 degrees centigrade for 12 h in 20 microliters in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM DTT, 60 mg/ml BSA, 10% (v/v) DMSO, and 40 U RNasin (Promega, Madison, WI, USA). 3' ligated product was purified from a 15% denaturing urea-polyacrylamide gel (National Diagnostics), and then ligated to 100 pmole of 5' RNA adapter (5'-GUU CAG AGU UCU ACA GUC CGA CGA UC-3') using T4 RNA ligase (Ambion, Austin, TX, USA) at room temperature for 6 h in 20 microliters in 50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, and 10% DMSO. The ligated product was purified from a 10% denaturing urea-polyacrylamide gel (National Diagnostics). Half the ligated product was used to synthesize cDNA using Superscript III (Invitrogen) and the reverse transcription primer (5'-CAA GCA GAA GAC GGC ATA CGA-3'), and half was used as a minus RT control. The small RNA library was amplified using forward (5'-AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-3') and reverse (5'-CAA GCA GAA GAC GGC ATA CGA-3') primers, and then purified from a NuSieve GTG agarose gel (Lonza, Basel, Switzerland). Purified libraries were sequenced using a Solexa Genome Analyzer (Illumina, San Diego, CA, USA). SRS212372 qinHet qinHet RNA-Seq TRANSCRIPTOMIC size fractionation 36 0 Application Read Forward 1 Illumina Genome Analyzer Solexa primary analysis Base Caller Solexa primary analysis Quality Scorer strategy piRNA-Seq RNA size 18-30 nt SRX078291 qinMut SRP007101 qin small RNA sequencing Total RNA was isolated from manually dissected ovaries from 2-4 day flies using mirVana (Ambion, Austin, TX, USA). The RNA was quantified by absorbance at 260 nm. 18-30 nt RNAs was selected from 100 micrograms total RNA by running a 15% denaturing urea-polyacrylamide gel (National Diagnostics, Atlanta, GA, USA). 2S rRNA was depleted by hybridization using a complementary, immobilized DNA oligonucleotide (5'-biotin-TCA ATG TCG ATA CAA CCC TCA ACC ATA TGT AGT CCA AGC A-3'). Briefly, for each library 400 µl MyOne Streptavidin C1 Dynabeads (Invitrogen) were washed twice in 0.5× SSC (1× SSC: 150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) at 4°C, then re-suspended in 400 µl 0.5× SSC. The beads were then loaded with 200 pmole DNA oligonucleotide at 4°C for 30 min, washed once in 0.5× SSC, re-suspended in 1 ml 0.5× SSC, and then warmed to 65°C for 5 min. Size-selected RNA (50 µg) was incubated at 80°C for 5 min, and then added to the pre-warmed beads and incubated at 50°C for 1 h. The beads were then removed by magnetic capture and the supernatant mixed with 3 volumes of ethanol, 0.3 M (f.c.) sodium acetate, pH 5.2, and 1 µl (20 µg/µl) glycogen (Roche, Indianapolis, IN, USA).100 pmole of 3' pre-adenylated adapter (5'-rAppTCG TAT GCC GTC TTC TGC TTG T/ddC/-3') was ligated to small RNAs using Mutant Rnl2_1-249_K227Q (17-23B) (Addgene, Cambridge, MA, USA) at 4 degrees centigrade for 12 h in 20 microliters in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM DTT, 60 mg/ml BSA, 10% (v/v) DMSO, and 40 U RNasin (Promega, Madison, WI, USA). 3' ligated product was purified from a 15% denaturing urea-polyacrylamide gel (National Diagnostics), and then ligated to 100 pmole of 5' RNA adapter (5'-GUU CAG AGU UCU ACA GUC CGA CGA UC-3') using T4 RNA ligase (Ambion, Austin, TX, USA) at room temperature for 6 h in 20 microliters in 50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, and 10% DMSO. The ligated product was purified from a 10% denaturing urea-polyacrylamide gel (National Diagnostics). Half the ligated product was used to synthesize cDNA using Superscript III (Invitrogen) and the reverse transcription primer (5'-CAA GCA GAA GAC GGC ATA CGA-3'), and half was used as a minus RT control. The small RNA library was amplified using forward (5'-AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA-3') and reverse (5'-CAA GCA GAA GAC GGC ATA CGA-3') primers, and then purified from a NuSieve GTG agarose gel (Lonza, Basel, Switzerland). Purified libraries were sequenced using a Solexa Genome Analyzer (Illumina, San Diego, CA, USA). SRS212373 qinMut qinMut RNA-Seq TRANSCRIPTOMIC size fractionation 36 0 Application Read Forward 1 Illumina Genome Analyzer Solexa primary analysis Base Caller Solexa primary analysis Quality Scorer strategy piRNA-Seq RNA size 18-30 nt SRX078292 Ago3_IP qinHet SRP007101 qin small RNA sequencing Ago3 IP in 400ul ovary lysate (5ug/ul), then following total small RNA library construction protocol. SRS212372 qinHet Ago3_IP qinHet RNA-Seq TRANSCRIPTOMIC size fractionation 36 0 Application Read Forward 1 Illumina Genome Analyzer Solexa primary analysis Base Caller Solexa primary analysis Quality Scorer strategy piRNA-Seq RNA size 18-30 nt SRX078293 Ago3_IP qinMut SRP007101 qin small RNA sequencing Ago3 IP in 400ul ovary lysate (5ug/ul), then following total small RNA library construction protocol. SRS212373 qinMut Ago3_IP qinMut RNA-Seq TRANSCRIPTOMIC size fractionation 36 0 Application Read Forward 1 Illumina Genome Analyzer Solexa primary analysis Base Caller Solexa primary analysis Quality Scorer strategy piRNA-Seq selection Ago3-IP RNA size 18-30 nt SRX078294 Aub_IP qinHet SRP007101 qin small RNA sequencing Aub IP in 400ul ovary lysate (5ug/ul), then following total small RNA library construction protocol. SRS212372 qinHet Aub_IP qinHet RNA-Seq TRANSCRIPTOMIC size fractionation 36 0 Application Read Forward 1 Illumina Genome Analyzer Solexa primary analysis Base Caller Solexa primary analysis Quality Scorer strategy piRNA-Seq RNA size 18-30 nt SRX078295 Aub_IP qinMut SRP007101 qin small RNA sequencing Aub IP in 400ul ovary lysate (5ug/ul), then following total small RNA library construction protocol. SRS212373 qinMut Aub_IP qinMut RNA-Seq TRANSCRIPTOMIC size fractionation 36 0 Application Read Forward 1 Illumina Genome Analyzer Solexa primary analysis Base Caller Solexa primary analysis Quality Scorer strategy piRNA-Seq selection Aub-IP RNA size 18-30 nt SRX078296 Piwi_IP qinHet SRP007101 qin small RNA sequencing Piwi IP in 400ul ovary lysate (5ug/ul), then following total small RNA library construction protocol. SRS212372 qinHet Piwi_IP qinHet RNA-Seq TRANSCRIPTOMIC size fractionation 36 0 Application Read Forward 1 Illumina Genome Analyzer Solexa primary analysis Base Caller Solexa primary analysis Quality Scorer strategy piRNA-Seq RNA size 18-30 nt SRX078297 Piwi_IP qinMut SRP007101 qin small RNA sequencing Piwi IP in 400ul ovary lysate (5ug/ul), then following total small RNA library construction protocol. SRS212373 qinMut Piwi_IP qinMut RNA-Seq TRANSCRIPTOMIC size fractionation 36 0 Application Read Forward 1 Illumina Genome Analyzer Solexa primary analysis Base Caller Solexa primary analysis Quality Scorer strategy piRNA-Seq selection Piwi-IP RNA size 18-30 nt