title: RNA-Seq of mus musculus
design: RNA quantification and qualification sequencing libraries were generated using NEBNext  Ultra  RNA Library Prep Kit for Illumina  (NEB, USA) following manufacturer s recommendations 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) the synthseis of double strand cDNA select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA) 3  l USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37 C for 15 min followed by 5 min at 95 C before PCR PCR was performed withPhusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system Illumia sequencing.