CUT&RUN of HOXA6 and HOXB6 in neuronal-derived human embryonic stem cells
CUT&RUN of HOXA6 and HOXB6 in neuronal-derived human embryonic stem cells
Cells were washed with PBS and trypsinized Cells were washed three times with PBS and twice with Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, protease inhibitors). Concanavalin A-coated (ConA) beads (Cell Signaling Technology) were activated according to manufacturer's protocol. Cells were then rotated for 10 minutes at room temperature with a 50% slurry of activated ConA beads. Next, 50ul Antibody Buffer (Wash Buffer, 0.1% Triton-X-100, 2 mM EDTA) was added to ConA bead-bound cells together with anti-HOXA6 (HPA004203, Atlas Antibodies), anti-HOXB6 (sc-166950, Santa Cruz), and IgG (011-0102, Rockland) antibodies and incubated for 4 hours with rotation. After antibody incubation, ConA bead-bound cells were washed twice with Tri-Wash Buffer (Wash Buffer, 0.1% Triton-X-100) and pA-MNase (gift from Alexander van Oudenaarden) was mixed into the sample at a final concentration of 70ng/ml for 1h at 4oC with rotation. ConA bead/pA-MNase-bound cells were washed twice with Tri-Wash Buffer. MNase was then activated by resuspending cells in ice-cold calcium incubation buffer (Tri-Wash Buffer, 2mM calcium chloride) and incubating on ice for 30 min. To stop the MNase reaction, EGTA-STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A, 50 μg/mL Glycogen) was added and incubated at 37°C for 20 min to degrade RNA and release cleaved chromatin into the supernatant. Cleaved chromatin was then subjected to reverse crosslinking with 0.1% SDS and 2ul Proteinase K (20mg/ml) at 65°C for 16 hours. Resultant DNA was purified using a QIAquick Gel Extraction Kit (QIAGEN).  Cut&Run samples were processed according to the protocol described by the Fazzio group.