GSM6732222: NRF2 SNU638; Homo sapiens; OTHER
Briefly，about 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  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 primary antibody incubated on a roating platform overnight at 4℃.  The primary antibody was removed using magnet stand. Sencondary antibody 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 digmed 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 min 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, 20μL DNA was mixed with 2μL of a universal i5 and auniquely barcoded i7 primer. A volume of 25 μL 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 by beads. The size distribution of libraries was determined and libraries were mixed to achieve equal representation as desired aiming for a final concentration as recommended by the manufacturer. Sequencing was performed in the Illumina Novaseq 6000 using 150bp paired-end following the manufacturer's instructions.