GSM7789960: mesc_rpb1-degron_IAA_Rad21_CUTTAG_rep1; Mus musculus; OTHER
Harvested cells were crosslinked with freshly made 1% PFA followed by 3 mM DSG, then cells were lysed and titrated to test the appropriate MNase concentration (fig. S2B). Before end repair, we added 50 μL 0.3% SDS and incubated at 62℃ for 10 min, then quenched by 50 μL 3% Triton X-100 and incubated at 37℃ for 15 min. Then nuclei were end repaired and biotin labeled, followed by in situ ligation with T4 ligase. Before DNA extraction, nuclei were incubated with exonuclease III (NEB, #M0206) to remove un-ligated ends (Micro-C). Cells were dissociated with trypsin and wash twice with 1x PBS at RT (CUT&TAG). Extracted DNA were either sonicated to 300 bp or select di-nucleosome fragments for library preparation (Micro-C). 0.1 million cells were used as input and processed with CUT&Tag kit (Vazyme, TD903) according to the manufacturer's instructions. RAD21 primary antibody (GeneTex, GTX106012) was used (CUT&TAG).