Drosophila pseudoobscura testes gene expression and small RNA sequencing

We dissected testes from 3-8 day old virgin males of D. pseudoobscura (strain MV25) reared at 18°C on Bloomington food. We used Trizol (Invitrogen) and GlycoBlue (Invitrogen) to extract and isolate total RNA. We used to Ribo-Zero to deplete ribosomal RNA from total RNA, and used Illumina's TruSeq Stranded Total RNA Library Preparation Kit to prepare stranded testis RNA libraries, which were sequenced on an Illumina HiSeq 4000 at 100 nt read length (paired-end). For small RNA data, we resolved 20 ug of total RNA on a 15% TBE-Urea gel (Invitrogen) and size selected 19-29 nt long RNA, and used Illumina's TruSeq Small RNA Library Preparation Kit to prepare small RNA libraries, which were sequenced on an Illumina HiSeq 4000 at 50 nt read length (single-end).