title: GSM8891289: BRD4S-2; Homo sapiens; OTHER
library_construction_protocol: CUT&Tag assay was performed as described previously with modifications (Hatice et al., 2019 ). Briefly, 100000 cells were washed twice gently with wash buffer (20 mM HEPES pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail). 10 μL Concanavalin A coated magnetic beads (Bangs Laboratories) were added per sample and incubated at RT for 10 min. Remove unbound supernatant and resuspended bead-bound cells with Primary Antibody Buﬀer (Mix 8 ul 0.5 M EDTA and 6.7 ul 30% BSA with 2 mL Dig-wash buffer) and a 1:50 dilution of primary antibody incubated on a rotating platform overnight at 4℃. The primary antibody was removed using magnet stand. Sencondary antibody (Goat Anti-Mouse IgG H&L (Cat. No. N270) or Goat Anti-Rabbit IgG H&L (Cat. No. N269)）was diluted 1:100 in dig wash buffer and cells were incubated at RT for 60 min. Cells were washed using the magnet stand three times in dig wash buffer. A 1:100 dilution of pA-Tn5 adapter complex was prepared in dig-300 buffer (0.01% Digitonin; 20 mM HEPES pH 7.5; 300 mM NaCl; 0.5 mM Spermidine; 1× Protease inhibitor cocktail) and incubated with cells at RT for 1 h. Cells were washed three times for 5 min in 1 mL dig-300 buffer. Then cells were resuspended in tagmentation buffer (10 mM MgCl2 in dig-300 buffer) and incubated at 37 °C for 1 h. DNA was purified using Tagment DNA Extract Beads. To amplify libraries, 21 μL DNA was mixed with 2 μL of a universal i5 and auniquely barcoded i7 primer. A volume of 25 μL NEBNext HiFi 2× PCR Master mix was added and mixed. The sample was placed in a Thermocycler with a heated lid using the following cycling conditions: 72 °C for 5 min (gap filling); 98 °C for 30 s; 14 cycles of 98 °C for 10 s and 63 °C for 30 s; final extension at 72 °C for 1 min and hold at 8 °C. libraried clean-up was performed XP beads (Beckman Counter).