Small RNAseq of Diceroprocta semicincta
RNA isolated using Roche High Pure miRNA kit following the 2-column protocol for <100nt RNAs. Libraries were prepared using ScriptminerTM Small RNA-Seq Library Preparation Kit from Epicenter. RT was done with an adapter specific primer and each library was subjected to 15 rounds of PCR using FailSafe PCR Enzyme Mix. PCR bands of approximate size 50-300nt were cut from an 8% polyacrylamide gel after staining with SYBRŽ Safe (Invitrogen), and visualized on a standard UV transilluminator. The gel was shredded using a 0.5 mL tube with needle holes in the bottom, and eluted with 300 uL 0.5 M ammonium acetate for 3.5 hours at 37°C. The liquid was separated from gel particles using a 0.22 micron sterile filter and DNA was purified by standard isopropanol precipitation.