title: RNA-Seq of Rattus norvegicus: adult male testis
design: The quality of purified total RNA provided will be checked on Agilent Technologies 2100Bioanalyzer using the Agilent RNA chip. Total RNA with RNA Integrity Number (RIN)value greater than or equal to 8 is considered good quality. Approximately, 4 g of totalRNA will be used to prepare the RNA-Seq library using the TruSeq RNA Sample Prep Kits(Illumina). The libraries will be prepared as per the kit protocol. In short, poly-A containingmRNA molecules will be purified using poly-T oligo-attached magnetic beads. Followingpurification, the mRNA will be fragmented into small pieces using divalent cations underelevated temperature. The cleaved RNA fragments will be then used to synthesize firststrand cDNA using reverse transcriptase and random primers followed by second strandcDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments will then go through an end repair process, followed by the addition of a single A base, and then ligation of the adapters. The products will be purified andenriched with PCR to create the final cDNA library. Bioanalyzer plots will be used at everystep to assess mRNA quality, enrichment success, fragmentation sizes, and final librarysizes. Both Qubit and qPCR will be used for measuring the quantity of the library beforesequencing.After the libraries are constructed they will be sequenced on HiSeq 2000/2500 toobtain 2x100bp PE (30 million high quality reads/sample). After the sequencing run,Illumina RNA-Seq data will be processed to generate FASTQ files and uploaded on theFTP server for download.