bulk RNA-sequencing of mouse embryonic fibroblast (MEF) reprogramming time course with control blue fluoresce protein (BFP) or Hic2 overexpression
MEFs with doxycycline (Dox)-inducible reprogramming cassette MKOS-ires-mOrange and a Nanog-GFP reporter were transduced with lentiviral Dox-inducible Ty1-BFP (control, BFP with a Ty1 tag at the N-terminus) or Ty1-Hic2 (HIC2 with a Ty1 tag at the N-terminus) expression vector with an MOI of 3. 1 day later, reprogramming was initiated by the administration of Dox. 2, 4, 6, 10 days later, mOrange expressing cells were flow sorted for RNA extraction. Nanog-GFP+ iPS cells were flow sorted from samples cultured for 15 days in the presence of Dox, and then cultured as a bulk population in the absence of Dox for 20 days before RNA extraction. In parallel, wild-type 129 MEFs with BFP or Hic2 overexpression for 3 days, E14Tg2a ES cells with BFP overexpression were also prepared as RNA-seq samples. Libraries were prepped with the NEB Ultra II stranded mRNA Library prep kit (NEB), and the sequence was carried out with NextSeq, 75SE.