GSM9566652: CUT&Run_SMARCA4_H23_SMARCA4; Homo sapiens; OTHER
CUT&RUN analysis was performed using the CUT&RUN Assay Kit (Cell Signaling Technology [CST]; 86652). Cells were harvested by trypsinization, and 5 × 105 cells were suspended in 1× Wash Buffer and bound to concanavalin A-coated beads. Cells were permeabilized with 100 μL of Antibody Binding Buffer containing digitonin and incubated at 4°C for 2 h with 2 μL of the following primary antibodies: anti-H3K4me3 (CST, 9751; RRID:AB_2616028), anti-H3K4me1 (CST, 5326; RRID:AB_10695860), anti-H3K27ac (CST, 8173; RRID:AB_2616015), anti-H3K27me3 (CST, 9733; RRID:AB_2616029), anti-SMARCA4 (Abcam, ab110641; RRID:AB_10860265), anti-CHD3 (Bethyl Laboratories, A301-219A; RRID:AB_890566). Cells were washed four times with Digitonin Buffer, incubated with pAG-MNase solution at 4°C for 1 h, and washed an additional four times. MNase was activated by adding 3 μL of CaCl2 solution at 4°C for 30 min, and the reaction was stopped by adding 1 × Stop Buffer. For input DNA preparation, 1 × 105 cells were treated with DNA Extraction Buffer containing RNase and Proteinase K, followed by incubation at 55°C for 1 hour. The genomic DNA was sonicated to yield fragments of 150–500 bp (40 cycles of 15 sec on/30 sec off, high setting) using a Bioruptor (M&S Instruments). Chromatin and input DNA were purified using DNA Purification Buffers and Spin Columns (CST; 14209). CUT&RUN-seq: Libraries were constructed using the ThruPLEX DNA-Seq Kit (Takara; R400674) and amplified for 16 cycles (denaturation at 98°C for 20 sec; annealing and extension at 72°C for 50 sec).