GSM7852751: Zic2rep2,CUTTag,H1WT; Homo sapiens; OTHER
hESCs were resuspended in 0.3 mL NE1 buffer (20 mM HEPES-KOH pH7.9, 10 mM KCl, 0.5 mM spermidine, 0.1% Triton X-100, 20% glycerol, plus protease inhibitor), then cell nuclei were obtained by centrifugation at 600 g for 5 min at 4°C. We adapted CUT&Tag direct protocol53 with some modifications. 0.5 μL primary antibody (anti-ZIC2, ABCAM, Cat#150404) and 0.5 μL normal IgG (Rabbit IgG, Millipore, Cat#12-370) was respectively diluted with 25 µL of Antibody Buffer (10 mM HEPES pH7.5, 150 mM NaCl, 0.5 mM spermidine, 2 mM EDTA, 0.1% BSA, plus protease inhibitor) and mixed with the above conjugated beads at 4°C overnight. After thoroughly aspirating the supernatant, 0.25 µL secondary antibody (Guinea Pig anti-Rabbit IgG, Antibodies-online, Cat#ABIN101961) in 25 µL Wash Buffer were added and incubated with the beads at room temperature for 1 hr. Extra antibodies were removed by washing the beads twice with 0.25 mL Wash Buffer. Then, custom-assembled pA–Tn5 transposases54 were loaded to the beads in 25 µL of 300-Wash Buffer (Wash Buffer with 300 mM NaCl) at room temperature for 1 hr. Following, unbound transposases were washed away with 300-Wash Buffer from the beads, and tagmentation was conducted in 50 µL of 300-Wash Buffer supplemented with 10 mM MgCl2 at 37 °C for 1 hr. After thoroughly aspirating the supernatant, the beads were washed once with TAPS buffer (10 mM TAPS pH8.5, 0.2 mM EDTA), then 5 µL SDS release buffer (10 mM TAPS pH8.5, 0.1% SDS) were added and incubated at 58 °C for 1 hr. Amplified tagmentated DNA were purified from the supernatant using 1.3X AMPure XP Beads, and subject to library sequencing.