GSM5516645: Sox2-Ctrl rep3; Mus musculus; RNA-Seq
Total RNA was prepared from the adult spinal cord  (P80) using QIAGEN RNeasy for lipid tissues with on-column DNase I digestion. The quality of RNA samples was determined by the Bioanalyzer 2100 system (Agilent Technologies). The RNA integrity number (RIN) of all our RNA samples used for RNA-seq was greater than 6.8. mRNA from the spinal cord was purified from total RNA using poly-T oligo-attached magnetic beads. The resulting mRNA was fragmented randomly by addition of fragmentation buffer. The cDNA library was prepared using the NEBNext Ultra Directional RNA Library Prep Kit (New England BioLabs). The first strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). The second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Double-stranded cDNA is purified using AMPure XP beads. Remaining overhangs of the purified double-stranded cDNA are converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure is ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Finally, the final library is gotten by PCR amplification and purification of PCR products by AMPure XP beads.