GSM5514759: SOX6_OL_sg1KO_rep1; Mus musculus; RNA-Seq
OPCs and immature oligodendrocyte cultures were lysed in TRIzol followed by purification and elution of RNA using phenol-chloroform extraction and the RNeasy Mini Kit. Specifically, RNA for defining in vitro OPC and immature oligodendrocyte expression profiles were generated from magnetically sorted OPCs and O1+ oligodendrocytes. For RNA isolated from sgNTC, sgSox6 g1, and sgSox6 g2 OPCs and immature oligodendrocytes, cells were plated into 6-well plates in OPC growth media and differentiation permissive media respectively for 60 hours at which point cells were lysed with TRIzol. Libraries were prepared following protocols from NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra RNA Library Prep Kit for Illumina. Samples were enriched for mRNA using oligo(dT) beads, which were fragmented randomly and used for cDNA generation and subsequent second-strand synthesis using a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H and DNA polymerase I. cDNA libraries then went through terminal repair, A-base ligation, adapter ligation, size selection, and PCR enrichment.