title: GSM8427640: Mock-infected  SMARCAKDBRD4-Rep3; Homo sapiens; OTHER
library_construction_protocol: Cells were trypsinized, quantitated and 4 x 106 cells were incubated on ice for 10 minutes in 1 ml of nuclear extraction buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 1x cOmplete proteinase inhibitor, 1x protein phosphatase inhibitor cocktail and 0.5 mM spermidine), the released nuclei were pelleted at 600x g, 5 min at 4 oC.  The nutated nuclei were resuspended in antibody buffer produced by diluting 5 g of anti-SMARCA4 Ab in 500 l of Triton X-100-free wash buffer (WB, 20 mM HEPES, pH7.5, 150 mM NaCl, 0.05% Triton X-100, 0.1% BSA, 1x cOmplete proteinase inhibitor, 1x protein phosphatase inhibitor cocktail and 0.5 mM spermidine), and incubated with nutation overnight at 4 oC.  The nuclei were washed and incubated at 4 oC for 1 h in 50 l of WB containing 2.5 l of EpiCypher pAG-MNase 20x stock (EpiCypher, NC). Chromatin cleavage was conducted by incubating the nuclei on ice for 1 h in 150 l of BSA-free WB containing 2 mM CaCl2. The cleavage was terminated by adding 150 l of stop buffer (300 mM NaCl, 20 mM EDTA, 4 mM EGTA and 0.5 ng of E. coli Spike-in DNA (EpiCypher, NC) per 150 l).  The samples nutated at 4 oC for 1 h, centrifuged at 16,000x g for 5 min at 4 oC and the supernatant was collected. The DNA fragments were phenol-chloroform extracted, glycogen precipitated, and dissolved in 20 l of 0.1x TE buffer. CUT&RUN DNA libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, MA) per the manufacturer's instruction, with modification in SPRI bead clearance of adaptor ligation and library amplification reactions to retain small-sized DNA fragments. The quality of the resultant DNA libraries was confirmed by Agilent TapeStation HS DNA assay (Agilent, Santa Clara, CA, USA), and paired-end Illumina NGS was carried out on NovaSeq 6000 with 5 million reads per sample. CUT&RUN DNA libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, MA) per the manufacturer's instruction, with modification in SPRI bead clearance of adaptor ligation and library amplification reactions to retain small-sized DNA fragments. The quality of the resultant DNA libraries was confirmed by Agilent TapeStation HS DNA assay (Agilent, Santa Clara, CA, USA), and paired-end Illumina NGS was carried out on NovaSeq 6000 with 5 million reads per sample.