GSM7656228: Rep_early_rep1_H3K4me1; Mus musculus; OTHER
The cells were lightly crosslinked by in 1 ml DMEM containing 0.1-0.15% formalin at RT for 1 min. Crosslinking was quenched by adding 50 ul 2.5 M glycine and rotated at RT for 10 minutes. The cells were resuspended in nuclear extraction buffer (NE buffer) (20 mM HEPES, pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 0.5 mM spermidine and EDTA-free protease inhibitors) (approximately 100 ul per 100,000 cells), and incubated on ice for 10 minutes. The extracted nuclei were washed once in NE buffer by centrifugation at 1,300 ×g at 4 °C for 3 minutes. The supernatant was removed, and the nuclei were resuspended in NE buffer, and aliquoted into an 8-well strip tube (100 ul per reaction). Then, 10 ul Concanavalin A-coated magnetic beads (EpiCypher) were added, and incubated at RT for 10 minutes. The nuclei/bead conjugate was bound to a magnet, the supernatant was removed, and were resuspended in 50 ul antibody reaction buffer (Wash150-T buffer with 5% digitonin and 0.5 M EDTA; the composition of Wash150-T buffer is 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, EDTA-free protease inhibitors and 0.05% Triton X-100), 0.5 ul antibody was added and incubated at 4°C overnight. After removal of the supernatant, the beads were resuspended in 50 ul Wash150-T buffer containing 0.5 ul secondary antibody, and incubated at RT for 30 minutes. Following two washes in 200 ul cold Wash150-T buffer, beads were resuspended in 50 ul Wash300-T buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM spermidine, EDTA-free protease inhibitors and 0.05% Triton X-100). Then, 2.5 ul pAG-Tn5 (EpiCypher) was added, and the samples were incubated at RT for 1 hour. While on the magnet, the supernatant was removed, and the samples were washed twice in 200 ul cold Wash300-T buffer. Following resuspension in 50 ul Tagmentation buffer (Wash300-T buffer with 10 mM MgCl2 (Sigma)), Tn5 digestion was performed at 37 °C for 1 hour. The supernatant was discarded, and the beads were washed once in in 50 ul TAPS buffer (10 mM TAPS (Boston BioProducts), pH 8.5, 0.2 mM EDTA). Tagmentation was quenched by adding 5 ul SDS release buffer (10 mM TAPS, pH 8.5, 0.1%SDS), mixed thoroughly by vortex and incubated at 58 °C for 1 hour. Then, 15 ul SDS quench buffer (0.67% Triton X-100) was added and mixed thoroughly by vortex. Then, 2 ul universal i5 primer (10 uM), 2 ul barcode i7 primer (10 uM), and 25 ul CUTANA® High Fidelity 2X PCR Master Mix for CUT&Tag (EpiCypher) was added, and PCR was performed (1 cycle of 20 °C for 5 min; 1 cycle of 72 °C for 5 min; 1 cycle of 98 °C for 45 seconds, 14-21 cycles of 98°C for 15 seconds and 60°C for 10 seconds; 72°C for 1 minute). The DNA was cleaned up with 1.3× AMPure XP beads (Beckman) following the standard procedure.