RNA-Seq of mus musculus: widetype testes
Testes were isolated, 1g RNA per sample was used for library preparation. Sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (New England Biolabs) following manufacturers recommendations and index codes were added 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 using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3 ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. The library fragments were purified with AMPure XP system (Beckman Coulter), size selected and ligated with adaptor using 3l USER Enzyme (New England Biolabs) at 37C for 15 min followed by 5 min at 95 C before PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified with AMPure XP system. After cluster generation, the library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated.
Sugp2-wildtype-1