GSM6856400
After washing in wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl (Sigma), 0.5 mM spermidine (Sigma) and EDTA-free protease inhibitors (Roche)) twice by centrifugation at 600 ×g at 4 °C for 3 minute The cells were resuspended in 150 ul wash buffer, 15 ul Concanavalin A-coated magnetic beads (EpiCypher) was added, and the cells/bead conjugate was bound to a magnet. After removal of the supernatant, the cells were resuspended in 50 ul antibody reaction buffer (wash buffer with 5% digitonin and 0.5 M EDTA), 0.5 ul antibody was added, and incubated at 4 °C overnight. Following two washes in 200 ul permeabilization buffer (wash buffer with 5% digitonin), beads were resuspended in 50 ul permeabilization buffer. Then, 2.5 ul pAG-MNase (EpiCypher) was added, and the samples were incubated at RT for 10 minutes. While on the magnet, the supernatant was removed, and the samples were washed twice in 200 ul cold permeabilization buffer. Following resuspension in 50 ul permeabilization buffer, 1 ul 100 mM CaCl2 was added to activate MNase, and MNase digestion was performed at 4 °C for 2 hours. The reaction was stopped by adding 33 ul STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 10 ug/ml RNase A, 50 ug/ml glycogen), and 1 ul E-Coli spike-in DNA (0.5 ng/ul) (EpiCypher) to each tube. After incubation at 37 °C for 10 minutes, beads were bound to the magnet, and the supernatant was transferred to a new tube. The DNA was cleaned up with the NEB Monarch kit (NEB), eluted in 12 ul elution buffer, and used as CUT&RUN DNA. Library preparation was performed using NEBNext Ultra II End Prep kit (NEB) with a slightly modified protocol. Briefly, after adaptor ligation, HA-Sox4 CUT&RUN DNA samples were purified with 1.75× AMPure XP beads (Beckman), while histone and IgG CUT&RUN samples were purified with 1.1× AMPure XP beads. 14-cycle PCR with index primers (NEB) was performed (initial denaturation: 1 cycle of 98 °C for 45 seconds; annealing/extension: 14 cycles of 98 °C for 15 seconds and 60 °C for 10 seconds; final extension: 72 °C for 1 minute). Finally, the library was purified with AMPure XP beads (for HA-Sox4 0.8× followed by 1.2×; for histone and isotype control 0.9×). For samples with adaptor contamination, further AMPure bead cleanup or gel extraction was performed.