KLF4 ChIP-seq at reprogramming 48 hours and Hic2, KLF4 ChIP-seq at reprogramming 48 hours with Hic2 overexpression
KLF4 ChIP-seq at reprogramming 48 hours and Hic2, KLF4 ChIP-seq at reprogramming 48 hours with Hic2 overexpression
Cas9 TNG MKOS MEFs (3.5 × 106 cells) were seeded onto 15-cm dishes (2 dishes in total) and cultured for 24h in MEF medium before infection with either a Tet-On Ty1-BFP or Ty1-Hic2-Ty1 lentiviruses at an MOI of 5. 48h later, cells were split across 5x 15-cm dishes and media was changed to reprogramming the following day for a duration of 48h. For Ty1-HIC2-Ty1 (Ty1 tag is linked in the C-term of HIC2 for ChIP experiments) ChIP, cells were crosslinked with 2mM of Disuccinimidyl Glutarate (DSG) for 45 min at room temperature (RT)  under constant agitation, before being cross-linked with 1% formaldehyde solution for 10 min at room temperatureRT. For KLF4 ChIPs, cells were only cross-linked with 1% formaldehyde solution for 10 min at room temperatureRT. Crosslinking was quenched by adding 120 mM glycine and incubating for 10 min at room temperature. Cells were collected using a scraper and pelleted by centrifugation at 1,350 × g for 5 min at 4 °C. The cross-linked pellets were washed 3 times with 10 ml of ice-cold PBS, and 3 to 4 aliquots of fixed cell pellets were made before flash freezing in liquid nitrogen and stored at –80 °C. Before nuclear extraction, 1 aliquot of cell pellets per ChIP was thawed on ice for 2–3 h and suspended in 10 ml filtered, ice-cold lysis buffer (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100 and 1 tablet of complete ultra-protease inhibitor cocktail (Roche)). The suspension was gently mixed on a rotating platform at 4 °C for 10 min. The cells were disrupted using a 7 ml glass-Dounce homogenizer (40 strokes) on ice. The nuclei were pelleted by centrifugation (1,350 × g for 5 min at 4 °C) and washed with 10 ml ice-cold wash buffer (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and 1 tablet of complete ultra-protease inhibitor cocktail (Roche)) for 10 min at 4 °C. The nuclei were collected by centrifugation and resuspended in 4 ml sonication buffer (10 mM Tis-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium-deoxycholate, 0.5% N-lauroylsarcosine and 1 tablet of complete ultra-protease inhibitor cocktail (Roche)). The resuspended nuclei were split into five aliquots in pre-chilled 1 ml milli-tubes containing AFA Fibre and sonicated using a Covaris-M220 focused-ultrasonicator (Covaris). Each milli-tube was sonicated for 10 min and kept on ice for 30 min per sonication cycle for a total of 4 cycles. Sonicated chromatin was transferred to Protein-Lobind tubes (Eppendorf). A total of 100 µl of 10% Triton X-100 was then added to each 1 ml sonicated chromatin to increase solubility. Chromatin samples were then centrifuged (20,000 × g at 4 °C for 10 min) and the supernatants were pooled into fresh tubes. A 50-µl aliquot of each pooled sample was analysed to check the fragment size distribution by Tapestation and to quantify the DNA content of the resulting sonicated chromatin using a Nanodrop spectrophotometer. Another 50-µl aliquot was retained to be used as input for ChIP analysis. The sonicated chromatin and the input sample were snap-frozen in liquid nitrogen and stored at -80 °C. For each ChIP analysischromatin precipitation, 30 µl Dyna Protein-G magnetic beads (Thermo Fisher Scientific) were washed three times with 1 ml blocking solution (0.5% w/v BSA in PBS/Tween-20). The beads were saturated with the appropriate antibody by adding 10 µg of Ty1 (Diagenode) or KLF4 (R&D) antibody and incubated on a rotating platform at 4 °C for at least 6 hours. Sonicated chromatin (60 µg) was then mixed with the antibody-saturated beads and incubated on a rotating platform at 4 °C overnight. The beads were transferred to a fresh tube, washed 5 times with 1 ml wash buffer (50 mM HEPES-KOH pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40 and 0.7% sodium-deoxycholate) and then washed once more with TE buffer (10 mM Tris-HCl pH 8, and 1 mM EDTA) containing 50 mM NaCl. The bound chromatin was eluted by incubating the beads in 210 µl EB (50 mM Tris-HCl pH 8, 10 mM EDTA and 1% SDS) at 65 °C for 30 min. The beads were pelleted by centrifugation at 16,000 × g for 1 min, and 200 µl supernatant containing the soluble chromatin was transferred to fresh tubes. The crosslinking was reversed by incubating the eluted chromatin and input DNA (in EB) at 65 °C for 16 h with shaking. An equal volume of TE (200 µl) was added to the eluted chromatin and input DNA to reduce the concentration of SDS. RNA in the samples was digested by adding 0.2 mg/ml RNase-A for 2 hours at 37 °C. Proteins were then digested by adding 0.2 mg/ml proteinase-K for 2 hours at 55 °C. ChIP DNA and input DNA were subsequently purified by phenol-chloroform extraction followed by ethanol precipitation and resuspended in EB buffer (100 mM Tris pH 8.0) before DNA concentrations were measured by Qubit Fluorometric Quantitation (Invitrogen, Thermo Fisher Scientific). DNA from ChIP assayschromatin immunoprecipitation and inputs were prepared using the NEBNext Ultra-II DNA Library Prep kit (New England Biolabs) according to the manufacturer's instructions. Each library was uniquely barcoded using NEBNext Multiplex Oligos for Illumina (Dual-Index Primers Set-1) (New England Biolabs). The quality of the DNA libraries was assessed using Agilent HS-DNA-Screen Tape (Agilent).