GSM7025902: WT_PolII_pS5_CutTag_rep2; Mus musculus; OTHER
Cells were washed in wash buffer 1 (20mM HEPES pH 7.5, 150mM NaCl, 0.5mM spermidine, Complete EDTA-free protease inhibitor cocktail). After washing, cells were bound to the Concanavalin A beads (Bangs Laboratories), followed by incubation with 1:50 primary antibody in wash buffer 1 supplemented with 0.05% digitonin, 2mM EDTA and 0.1% BSA at 4°C overnight. The next day, cells were incubated with 1:100 secondary antibody in wash buffer 1 supplemented with 0.05% digitonin at room temperature for 1 h on a rotator. After washing, cells were mixed with 1:200 pA-Tn5 (124601, Addgene; purified in-house) in wash buffer 2 (20mM HEPES pH 7.5, 300mM NaCl, 0.5mM spermidine, Complete EDTA-free protease inhibitor cocktail) plus 0.01% digitonin at room temperature for 1 h on a rotator. Cells were then washed twice, followed by tagmentation in wash buffer 2 plus 0.01% digitonin and 10mM MgCl2 at 37°C for 1 h. DNA was solubilized by adding 16.7mM EDTA, 0.1% SDS and 0.167mg/mL Proteinase K to each sample and incubating at 55°C for 1 h. DNA was phenol-chloroform extracted. Libraries were indexed and amplified using the NEBNext High-Fidelity 2x PCR Master Mix (M0541S, NEB) and ATAC-seq primers, followed by purification using AMPure XP beads.