title: NextSeq 500 sequencing; RNA-seq of in vitro reprogrammed mouse pericytes to oligodendrocytes
design: RNA-seq of in vitro reprogrammed mouse pericytes to oligodendrocytes
library_construction_protocol: Pericytes (PCs) were cultured in pericyte medium. Mouse embryonic fibroblasts (MEFs) were cultured in MEF medium. Oligodendrocytes (pOPCs and iOPCs) were cultured in OPC medium. The cells were cultured in a humidified incubator at 37C under 5% CO2.  To induce differentiation of MEFs or PCs into oligodendrocytes, PC and MEFs were transduced with virues containing Olig2/Sox10 (2F-pOPCs) or Olig2/Sox10/Nkx6.2 (3F-pOPCs). RNAs were extracted from 1 Mio. cells using RNeasy Kit (Qiagen), followed by DNase digestion using TURBO DNase (Thermo Fisher).  RNA was quantified using Nanodrop and RIN score quality assessment was determine using Bioanalyzer. PolyA-mRNA was isolated from 1 microg of total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) before sequencing library preparation. mRNAs were isolated from 1 microg of total RNA using a PolyA selection kit (NEB) and sequencing libraries were prepared following instructions from NEBs Ultra Library Preparation Kit for Illumina. All samples were barcoded, amplified in 12x PCR cycles, pooled and sequenced on a HiSeq2500 Sequencer (Illumina) using a 50 bp single-end run.