title: smallRNA-seq of Gryllus bimaculatus brains - red_RNAi replicate 1
design: Unmated adult male crickets within one week of their final molt were injected with 2 L of dsRNA (Gb-oskar, Gb-piwi and dsRed dsRNA) through a hole pierced in the median ocellus (Matsumoto et al. 2003) using a 10 L syringe fitted with 26S gauge tip (WPI, Tokyo, Japan; Hamilton Inc., Nevada, USA). At 48h post RNAi injections, brains were dissected in ice-cold 1x PBS, and transferred into Trizol, following which total RNA was extracted from them following manufacturers protocols. Next, RNA was size selected for 18-30nt size range after denaturing PAGE. 2S rRNA specific oligo was used for 2S rRNA depletion. The small RNAs were ligated at 3 and 5 ends by the respective adapters and purified by denaturing PAGE after each ligation. PCR was performed after reverse transcription. The PCR product was gel purified from agarose gel to obtain the final library. The libraries were sequenced using Illumina NextSeq500.