title: RNA-Seq of mus musculus: adult male testis
design: Total RNA was used as input material for the RNA sample preparations. Sequencinglibraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA,Catalog #: E7530L) following manufacturers recommendations and index codes wereadded to attribute sequences to each sample.Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads.Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized usingrandom hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Second strandcDNA synthesis was subsequently performed using DNA Polymerase I and RNase H.Remaining overhangs were converted into blunt ends via exonuclease/polymeraseactivities. After adenylation of 3 ends of DNA fragments, NEB Next Adaptor with hairpinloop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XPsystem (Beverly, USA). Then 3 L USER Enzyme (NEB, USA) was used with size selected,adaptor-ligated cDNA at 37C for 15 min followed by 5 min at 95 C before PCR. ThenPCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primersand Index (X) Primer. At last, PCR products were purified (AMPure XP system) and libraryquality was assessed on the Agilent 5400 systemAgilentUSA and quantified by QPCR(1.5 nM).The qualified libraries were pooled and sequenced on Illumina platforms with PE150strategy in Novogene Bioinformatics Technology Co., Ltd (Beijing, China), according toeffective library concentration and data amount required.