RNA-Seq sRNA Ictalurus furcatus male testes tissue
An initial amount of 3 μg total RNA per sample (N = 8 with 4 from the high velocity group, and 4 from the low velocity group) was used to construct the sRNA library, via NEBNext® Multiplex Small RNA Library Prep Set. cDNA was synthesized using M-MuLV Reverse transcriptase (RNase H-), and PCR amplification was done using LongAMP Taq 2X Master Max, SR primer for Ilumina and index (X) primer. The products of PCR amplification were purified on 8% polyacrylamide gel at 100V for 80 min, followed by the recovery and elution of DNA fragments between 140 bp and 160 bp. Library quality was assessed using an Agilent Bioanalyzer 2100, using DNA high sensitivity chips. Index-coded samples were clustered using a cBot Cluster Generation System with a TruSeq SR Cluster Kit v3-cBot-HS. Sequencing was then run on an Illumina sequencing instrument (Novoseq 6000, HWI-ST1276, Illumia, San Diego CA, USA) where 50 bp single-end reads were generated.