GSM8510896: CG_UNTREATED_D13_REP3; Homo sapiens; ATAC-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. Nuclei were prepared from 100-250k cells frozen cells by gentle lysis in 100uL isolation buffer (10uL of Tris-HCl pH 7.5 1M, 2uL of NaCl 5M, 3uL of MgCl2 1M, BSA 1%, Tween-20 0.1%, NP40 0.05% Digitonin 0.005%, 1uL of DTT 1M, 25uL of RNase inhibitor 40U/uL in 843uL of water) for 5min on ice. Nuclei were washed with 400uL of wash buffer (250uL of Tris-HCl pH 7.5 1M, 50uL of NaCl 5M, 75uL of MgCl2 1M, BSA 1%, Tween-20 0.1%, 25uL of DTT 1M, 625uL of RNase inhibitor 40U/uL in 20.925mL of water), strained (40um), and centrifuge for 5 min (500g, 4 °C). Nuclei were resuspended in nuclei buffer (50uL of 20x nuclei buffer, 1uL of 1M DTT, BSA 1%, 824uL water). Tagmentation was performed for 50,000 targeted nuclei using the Illumina Tagment DNA TDE1 Enzyme and Buffer Kits #15027865. Post-tagmentation DNA fragment recovery was performed using a Guanidium-based method. Briefly, 25uL of guanidinium hydrochloride solution 8 M, pH 8.5 (MBS) was added for 5 minutes, followed by 120uL of binding solution (BS: 20% PEG 8000, 2.5M NaCL, 10mM Tris-HCl pH8, 1mM EDTA, 0,05% Tween 20) and 30uL of MBSpure beads. After 10 minutes of incubation, fragments were purified on a magnetic stand and washed twice with 150uL of 80% EtOH.