GSM8510839: CG_23_HA_IP_R1; Homo sapiens; ChIP-Seq
For differentiation, 2000 hiPSCs were seeded as clumps per well of a Matrigel-coated 12-well plate and 3 days later normoxic cell culture with STEMdiff hematopoietic differentiation basal medium with supplement A (containing BMP4, FGF2, VEGFA) for the first day with 3 µM CHIR99021 glycogen synthase kinase 3 inhibitor (Sigma-Aldrich) and another 2 days without. Next, cells were cultured for 9-10 days in differentiation medium with supplement B (containing BMP4, FGF2, VEGFA, SCF, FLT3L, TPO) with half media changes every 2–3 days. For CG protein degradation dTAG-13 compound (100 nM final; kindly provided by Nathanael Gray, Dana-Farber Cancer Institute, Boston, MA, USA; CG-negative isogenic control) was added from differentiation day 2 onwards. Approx. 10^7 CG-expressing long-term or M-07e cells were fixed for 10 min at RT with 1% methanol-free formaldehyde in DPBS, and chromatin was prepared with the truChIP chromatin shearing kit according to NEXSON protocol 66 by ultrasonication for 20 min at 75 W peak incidence power, 10% duty factor, 200 cycles per burst and 7°C (Covaris M220). Immunoprecipation was performed with chromatin of 3-4 * 106 cells, 3 µg HA, CBFA2T3, or control normal mouse immunoglobulin G, and Protein G dynabeads (Invitrogen). Libraries were prepared from input and immunoprecipitated DNA using the NEBnext Ultra II kit (New England Biolabs, E7645L) and sequenced on an Illumina NovaSeq (either 2x150 cycles in paired-end [CG_22_HA_IP_R1.1, CG_22_INPUT_R1.1, sub_M07E_CBFA2T3_IP, sub_M07E_INPUT] or 1x100 cycles in single-end [all other samples] setup) by the Vienna BioCenter Core Facilities (VBCF).