GSM8795427: D7,RUNX1,Rosa,14; Mus musculus; OTHER
30,000-60,000 GFP-positive cells were sorted by FACS and the CUT&RUN sequencing was performed as previously described44 with minor modifications. 10% Kasumi-1 cells were added as spike-in. Concanavalin-A (ConA) beads were activated by washed twice using activation buffer (20 mM HEPES–KOH pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). Sorted cells were resuspended in room temperature (RT) wash buffer (20 mM HEPES–KOH pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitor cocktail) and added into ConA beads. After incubating 10 min in RT, ConA-beads-bound cells were incubated overnight at 4 °C in antibody buffer (wash buffer supplemented with 0.01% digitonin and 2 mM EDTA) and antibody. The following primary antibodies were used: SMARCA5 (EpiCypher, 13-2007, 2.5 μl) and CTCF (CST, 3418S, 1μl). After antibody incubation, ConA-beads-bound nuclei were washed once with Digitonin buffer (wash buffer supplemented with 0.01% digitonin) then resuspended and incubated at 4 °C for 1 h in Digitonin buffer and 0.1 μl pAG-MNase (a gift from Dr. Wei Xie, Tsinghua University). ConA-beads-bound nuclei were then washed twice with Digitonin buffer and resuspended in 98 μl of Digitonin buffer and incubated on ice for 5 min. Then, 2 μl 100 mM CaCl2 was added and mixed gently to each 100 μl ConA-bound nuclei. The reaction was then incubated at 4 °C for 30 min. The reaction was stopped by addition of 100 μl 2× stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% Digitonin and 50 μg/ml RNaseA, 50 μg/ml Glycogen) and incubated at 37 °C for 10 min. All buffers mentioned above were filtered with 0.22 μm filter before use. After incubation, ConA-bound nuclei were captured using a magnet and supernatant containing CUT&RUN DNA fragments were collected. Supernatant was incubated at 70 °C for 10 min with 2 μl 10% sodium dodecyl sulfate, 3 μL 10 mg/ml proteinase K and 2 μL 20ng/μL carrier RNA (Qiagen, 1068337). DNA was purified using PCI reagent (Solarbio, P1012) and overnight ethanol precipitation with glycogen at -20 °C. DNA was resuspended in water and repaired to 5´phosphorylated and 3´ dA-tailed DNA with NEBNext® Ultra™ II End Repair/dA-Tailing Module (NEB, E7546S). Barcodes (BIOO Scientific, 514153) were ligated with Quick Ligation kit (NEB, M2200). After size selection, 16 cycles of PCR were used to amplify the library. PCR-amplified libraries were purified using KAPA beads (Kapa biosystems) and sent for sequencing.