Ab191401

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.

Western blot was performed as described(Xu et al., 2012 (link)). Briefly, samples were boiled in 1× SDS loading buffer to denature all proteins and separated with 13% or gradient SDS-PAGE gels. Proteins were then transferred to PVDF membrane with a standard wet transfer system at 2.5 mA/cm² for 2 hr. Membranes were blocked with 5% nonfat milk for 1 hr and then incubated with primary antibodies for 1 hr at room temperature or overnight at cold room with shaking. Excess antibodies were washed with TBS-T (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% Tween 20) for 3 times and HRP-conjugated secondary antibodies were incubated for 30 min at room temperature. After 3 washes with TBS-T, the membranes were developed with Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0500). The following antibodies were used: M2-Flag (F1804, Sigma-Aldrich), BCL11A (ab19487 and ab191401, Abcam), and GAPDH (sc-25778, Santa Cruz Biotechnology). All antibodies were used at 1:1,000 dilutions in TBS-T.