Sequencing of small non-coding RNA and mRNA in Drosophila testes

Ago immunoprecipitation and sRNA extraction: Six hundred testes of cyp40 heterozygous control (cyp40KO/TM6) or transheterozygous mutant (cyp40KO/Df) flies of less than 3 days old were dissected and homogenized using plastic pestle. For FLAG-HA-Ago2 (FH-Ago2) IP, anti-DYKDDDK antibody-conjugated magnet beads (MBL) were incubated for 1h on ice. For Ago1 IP, anti-Ago1 antibody (ab5070, 1:50) for 1h on ice, then further incubated with protein A magnet beads (Milipore) for another 1h on ice. The beads-bound sRNAs were extracted with TRizol LS eagent (Thermo) following the manufacture protocol, and precipitated in the presence of 50%(v/v) isopropanol and 20 micro gram of glycogen overnight at -20C. The sRNA-seq libraries were constructed with NEB Next Multiplex Small RNA Library Prep Set for Illumina (NEB). After agarose gel electrophoresis, the fragments containing target sRNAs were extracted and sequenced by Illumina HiSeq2500. 3 prime adaptor sequence (5-AGATCGGAAGAGCACACGTCT-3). Transcriptome analysis: RNA (>200nt) was extracted from 400 testes of less than 3 days old flies for each genotype using miRNeasy Mini column (QIAGEN). For the first replicate of heterozygous control (cyp40KO/TM6) and cyp40 mutant (cyp40KO/Df), the extracted RNA was treated with DNase I (NEB) and rRNA fragments were removed by Ribo-Zero rRNA removal kit (Illumina). The libraries were constructed by using TruSeq standard mRNA Library Prep Kit (Illumina), and sequenced by Illumina HiSeq3000. For the remaining samples, purified >200-nt RNA was passed through oligo dT column, and libraries were constructed using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB), and sequenced by Illumina HiSeq X.