design: bulk RNA-sequencing of mouse embryonic fibroblast (MEF) reprogramming time course with control blue fluoresce protein (BFP) or Hic2 overexpression
library_construction_protocol: For control and Hic2 overexpressing MEF samples, 1 × 105 Cas9 TNG MKOS MEFs were transduced with either Ty1-BFP (control, BFP tagged with a Ty1 tag at the N-term end ) or Ty1-Hic2 (HIC2 tagged with a Ty1 tag at the N-term end) lentiviruses at an MOI of 3 with 8 µg/ml polybrene (Merck-Millipore) for 4 h. After 3 days in culture in MEF media, the cells were harvested and FACS-sorted to exclude dead cells with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (ThermoFisher Scientific, Dilution: 1/1500). For reprogramming samples, 0.5 × 104 Cas9 TNG MKOS MEFs were mixed with 9.5 × 104 WT MEFs (129 strain) and seeded in gelatin-coated wells of 6-well plates. Cells were transduced with either Ty1-BFP or Ty1-Hic2 lentivirus at an MOI of 3 with 8 µg/ml polybrene (Merck-Millipore) for 4h, before being recovered for 24 h in MEF media. Reprogramming was initiated by the addition of a reprogramming medium. Cells were harvested at day 2, day 4, day 6 and day 10 of reprogramming, respectively, and a maximum of 1 × 105 of mOrange+ OSKM and Nanog-GFP+ expressing cells were sorted with the FACS AriaII (BD Biosciences) per sample. Additionally, iPSCs were generated from Cas9 TNG MKOS MEFs transduced with a tet-inducible version of the Ty1-BFP and Ty1-Hic2 lentiviral particles Nanog-GFP+ iPSCs were harvested at day 15, and sorted with the FACS AriaII (BD Biosciences) and put in culture in ESC medium for 20 days in absence of doxycycline.   All cells were homogenized with the QIAshredder kit (Qiagen) and total RNA was extracted from all samples using the RNeasy Plus Micro Kit (Qiagen).  Libraries were prepped with the NEB Ultra II stranded mRNA Library prep kit (NEB).