title: GSM5366195: Sample 27: HT1376-TetOn-ARID1A_DMSO_without Dox_H3K27ac_R1; Homo sapiens; OTHER
library_construction_protocol: Cells were fixed by adding formaldehyde to final concentration of 0.1%. Fixation was done for 1 min at room temperature. Crosslinking was stopped by adding glycine to a final concentration of 125 mM. The fixed cells were snap-frozen and shipped by Epicypher (Durham, NC) for CUT&RUN processing. 0.5x106 fixed cells were washed twice in 100 uL Wash Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Protease Inhibitors) before resuspending in 100 uL Wash Buffer and transfer to tubes containing 10 uL of activated ConA bead (Epicypher #21-1401). The ConA beads absorbed cells were resuspend in 50 uL Antibody Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.02% Digitonin, 2 mM EDTA,  1x Protease Inhibitors) containing 0.5 ug of antibodies recognizing ARID1A (BETHYL A301-041A), SMARCA4 (BETHYL A300-813A), H3K4me3 (Epicypher #13-0041), H3K27me3 (Epicypher #13-0030), H3K27ac (Epicypher #13-0045) or IgG (Epicypher #13-0041). The beads and antibodies were incubated overnight at 4C before washing with 250 uL Digitonin Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.01% Digitonin, 1x Protease Inhibitors) twice. The beads were incubated with 50 uL cold Digitonin Buffer with 2.5 uL CUTANA pAG-MNase (Epicypher # 15-1016) for 10 min at room temperature before washing with 250 uL cold Digitonin Buffer twice. The washed beads were incubated with 50 uL Digitonin Buffer with 1 uL 100 mM CaCl2 for 2 hr at 4C.  The released DNA were purified by NEB Monarch DNA Cleanup Kit (NEB T1030S) and subjected for library preparation using NEB Ultra II Library Kit (NEB E7645S) following the manufacturer's instructions. Prior to sequencing, library size and concentration were assessed by Agilent High Sensitivity DNA ChIP (Agilent 5067-4626) on 2100 Bioanalyser (Agilent). Sequencing was performed with an Illumina MiniSeq or NextSeq 500 platforms to generate 3-5 million paired-end reads.