Transcriptional analysis of live and hybridized iPSCs

Mouse induced pluripotent stem cells (iPSCs) were derived from embryonic fibroblasts by overexpressing the Yamanaka factors Oct4, Sox2, Klf4 and c-Myc, and then grown in standard 2i/serum media conditions. Hybridized iPSCs were fixed with 4% paraformaldehyde; permeabilized in 70% ethanol for over 12 hours; incubated for 12 hours at 30C in an RNA preserving hybridization buffer (300 mM Sodium chloride, 30mM Sodium citrate, 2.1M Ammonium sulfate, 25% formamide, 10 mM EDTA, 1 mg/ml E. Coli tRNA, 500 μg/ml BSA); and reverse cross-linked for 1 hour at 50C with Sodium dodecyl sulfate (SDS) and Proteinase K (100 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.5% SDS, 500 μg/ml Proteinase K). RNA was extracted and compared for live and hybridized iPSCs (1 sample each).