GSM7025970: 4CBS_CUT&Tag_ab4729; Mus musculus; OTHER
To prepare nuclei, mESC were dissociated with Accutase (Innovative Cell Technologies, AT104), collected by centrifugation, washed twice with PBS (Gibco, 10010023) then resuspended in cold nuclei permeabilization buffer (10 mM Tris-HCl pH 7.4 (Sigma, T4661), 10 mM NaCl (Sigma, S7653), 3 mM MgCl2 (Sigma, 63069), 1×Protease Inhibitor (Roche, 05056489001), 0.5 U µl−1 RNase OUT (Invitrogen, 10777-019), 0.5 U µl−1 SUPERase Inhibitor (Invitrogen, AM2694), 0.1% IGEPAL CA630 (Sigma, I8896) and 0.02% Digitonin (Sigma, Cat#D141)) for 1 minute. Nuclei were counted using a BioRad TC20 cell counter with 0.4% Trypan Blue (Gibco, 15250061) staining. For each experiment, around 0.5 million nuclei were aliquoted, and centrifuged for 10 min at 1,000g, 4 °C before proceeding to Bulk CUT&Tag. Tagmentated products are extracted using QIAGEN MiniElute after CUT&Tag, and directly amplified with indexed primers. Size selection using SPRI Beads is performed with amplified products to obtain the final sequencing library (150-800 bp)