title: GSM6234182: CUT&TAG_G; Homo sapiens; OTHER
library_construction_protocol: NK cells were separated from NG-BM and G-BM using FACS 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). 10μL Concanavalin A coated magnetic beads (Bangs Laboratories) were added per sample and incubated at RT for 10min. Remove unbound supernatant and resuspended bead-bound cells with dig wash buffer (20mM HEPES pH 7.5; 150mM NaCl; 0.5mM Spermidine; 1× Protease inhibitor cocktail; 0.05% Digitonin; 2mM EDTA) and a 1:50 dilution of GR (G-5,sc-393232,SantaCruz) or IgG control antibody (Millipore cat.no. 12-371) incubated on a roating platform overnight at 4°C. The primary antibody was removed using magnet stand. Secondary antibody (abcam,ab6709) was diluted 1:100 in dig wash buffer and cells were incubated at RT for 60 min. Cells were washed using the magnet stand 2-3 times in dig wash buffer . A 1:100 dilution of pA-Tn5 adapter complex was prepared in Dig-med buffer (0.01% Digitonin; 20mM HEPES pH 7.5; 300mM NaCl; 0.5mM Spermidine; 1× Protease inhibitor cocktail) and incubated with cells at RT for 1h. Cells were washed 2-3× for 5 mins in 1 mL Dig-med buffer. Then cells were resuspended in tagmentation buffer (10mM MgCl2 in Dig-med Buffer) and incubated at 37 °C for 1 h. DNA was purified using phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. To amplify libraries, 21μL DNA was mixed with 2μL of a universal i5 and a uniquely barcoded i7 primer. A volume of 25μL 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. Libraries clean-up was performed using XP beads (Beckman Counter).