GSM5964215: CUTnRUN_BRG1_IpmkKD_SA_rep2; Mus musculus; OTHER
CUT&RUN assays were performed as previously described (Meers et al., 2019; Skene and Henikoff, 2017), with minor modification. Briefly, 4 million mESCs were harvested and washed with 1.5 ml Wash buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine) three times. Cells were bound to activated Concanavalin A-coated magnetic beads (at 25°C for 10 min on a nutator), then permeabilized with Antibody buffer (Wash buffer containing 0.05% Digitonin and 4 mM EDTA). The bead-cell slurry was incubated with 3 μl relevant antibody (see below) in a 150 μl volume at 25°C for 2 hours on a nutator. After two washes in 1 ml Dig-wash buffer (Wash buffer containing 0.05% Digitonin), beads were resuspended in 150 μl pAG/MNase and incubated at 4°C for 1 hour on a nutator. After two washes in 1 ml Dig-wash buffer, beads were gently vortexed with 100 μl Dig-wash buffer. Tubes were chilled to 0°C for 5min and ice-cold 2.2 mM CaCl2 was added while gently vortexing. Tubes were immediately placed on ice and incubated at 4°C for 1 hour on a nutator, followed by addition of 100 μl 2xSTOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 0.1 mg/ml RNase A, 50 μg/ml glycogen) and incubated at 37°C for 30min on a nutator. Beads were placed on a magnet stand and the liquid was removed to a fresh tube, followed by addition of 2 μl 10% SDS and 2.5 μl proteinase K (20mg/ml) and incubated at 50°C for 1 hour. DNA was extracted using phenol chloroform as described at https://www.protocols.io/view/cut-amp-run-targeted-in-situ-genome-wide-profiling-zcpf2vn. CUT&RUN libraries were prepared using either a Accel-NGS 2S Plus DNA Library Kit (21024, SWIFT), according to the manufacturer's guidelines. The libraries were then sequenced using an Illumina Novaseq 6000 platform. The libraries were generated from two sets of biological replicates. CUT&RUN libraries for genome-wide sequencing were prepared using either a Accel-NGS 2S Plus DNA Library Kit (21024, SWIFT).