RNA-seq of NSCs derived from hiPSCs-B
Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) accordingto the manufacturers protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer(Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gelelectrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo(dT)beads, while prokaryotic mRNA was enriched by removing rRNA by Ribo-ZeroTM MagneticKit (Epicentre, Madison, WI, USA). Then the enriched mRNA was fragmented into shortfragments using fragmentation buffer and reverse transcripted into cDNA with randomprimers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP andbuffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit(Qiagen,Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencingadapters. The ligation products were size selected by agarose gel electrophoresis, PCRamplified, and sequenced using Illumina HiSeq2500 by Gene Denovo Biotechnology Co.(Guangzhou, China).