Small RNA Sequencing from Drosophila Ovaries

For small RNA sequencing, two replicates per strain were prepared. Small RNA was isolated from 50 pairs of ovaries using HiTrap Q HP anion exchange columns (Cytiva, Velizy-Villacoublay, France) as described in [37 (link)], and the eluate was run on a 10% TBE urea gel (Thermo Fisher Scientific). Small RNA size selection (18–50 bp) was performed on gel at the sequencing facility. Quality was checked with the Bioanalyzer small RNA kit (Agilent, Santa Clara, CA, USA). Library construction was performed using the TruSeq Small RNA Library kit (Illumina, San Diego, CA, USA) and sequenced (1 × 50 single reads) on an Illumina HiSeq 4000 at the IGBMC Microarray and Sequencing facility. Adapter sequences were removed using cutadatp [38 (link)]. Size selection was then performed using PRINSEQ lite version 0.20.4 [39 (link)]. All subsequent analyses were built upon small RNA counts after normalization according to the miRNA amounts, as described in [34 (link)].