Large RNA-seq of Rattus norvegicus: 1 year F3 sperm
Total RNA was used to construct whole transcriptome libraries using the KAPA Stranded RNA-seq Library Preparation kit with RiboErase, according to the manufacturers instructions, with some modifications. The adaptor and barcodes used were from NEBNext Multiplex Oligos for Illumina. Prior to PCR amplification, libraries were incubated at 37C for 20 minutes with the USER enzyme (NEB). PCR cycle number was determined using qPCR with the KAPA RealTime Library Amplification kit before final amplification. Size selection (300-700 bp) was performed using Agencourt AMPure XP beads (Beckman Coulter). Quality control was performed using Agilent DNA High Sensitivity chips (Agilent) and Qubit dsDNA high sensitivity assay (ThermoFisher). Libraries were pooled and loaded onto an Illumina NovaSeq S1 2x50 chip, and sequenced on an Illumina NovaSeq 6000 sequencer.