Ab11852

CUT&RUN experiments were carried out as described (Skene and Henikoff, 2017 ) with modifications. Briefly, nuclei from 2×10⁶ cells were isolated with NE buffer (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 0.5 mM Spermidine, 0.1% Triton X-100, 20% Glycerol and 1× protease inhibitor cocktails from Sigma), captured with BioMag®Plus Concanavalin A (Polysciences) and incubated with primary antibody for 2 hours. After washing away unbound antibody with wash buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA and 1× protease inhibitor cocktails from Sigma), protein A-MNase was added at a 1:1000 ratio and incubated for 1 hour. The nuclei were washed again and placed in a 0°C metal block. To activate protein A-MNase, CaCl₂ was added to a final concentration of 2 mM. The reaction was carried out for different time courses and stopped by addition of equal volume of 2×STOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A and 40 μg/mL glycogen). The protein-DNA complex was released by centrifugation and then digested by proteinase K at 50°C overnight. DNA was extracted by ethanol precipitation, followed by Qubit fluorometer and bioanalyzer quality control. Protein A-MNase (batch 5) was kindly provided by Dr. Steve Henikoff. The antibodies used were: GATA1, ab11852, abcam; CTCF, 07-729, Millipore; BCL11A, ab191401, abcam; normal rabbit IgG, 12-370, Millipore.

ChIP-sequencing was obtained from primary human erythroblasts from CD34⁺ HSPCs subject to erythroid differentiation conditions using a GATA1 antibody (ab11852; Abcam), TAL1 antibody (clone C-21; Santa Cruz), and H3K27Ac antibody (Abcam, ab4729). ChIP-qPCR data was obtained from HUDEP-2 cells six days after lentiviral transduction with CRISPR/Cas9 reagents.