title: GSM9538441: NUGC4,H3K27me3,KO,rep1; Homo sapiens; OTHER
library_construction_protocol: Cells were trypsinized and permeabilized in ONMI buffer (10 mM Tris-HCl pH 7.5, 0.1% Tween20, 0.1% NP40, 0.01% digitonin, 3 mM MgCl2, 10 mM NaCl) for 5 min at 4°C. Approximately 100,000 permeabilized cells were resuspended in 50 µl buffer Med1 (2 mM EDTA, 2% BSA, 300 mM NaCl, 20 mM HEPES pH 7.5, 0.01% Digitonin, 0.5 mM spermidine, 0.01% NP40, 1x Protease Inhibitor Cocktail) containing 1 µl H3K27me3 antibody (Abcam, ab192985) and 1 µl pA-Tn5 transposase, and incubated with gentle rotation at 4°C overnight. Excess antibody and pA-Tn5 were removed by three washes with buffer Med2 (2% BSA, 300 mM NaCl, 20 mM HEPES pH 7.5, 0.01% Digitonin, 0.5 mM spermidine, 0.01% NP40, 1x Protease Inhibitor). Tagmentation was initiated by addition of 10 mM MgCl2 to Med2, followed by incubation at 37°C for 1 h. Fragmented DNA was purified using QIAquick PCR Purification Kit (Qiagen, #28106). Purified tagmented DNA was amplified using NEBNext High-Fidelity 2X PCR Master Mix (NEB, #M0541L). Libraries were purified, quality-checked, and size-selected. Paired-end 150 bp sequencing was performed on an Illumina NovaSeq X Plus platform.