GSM7774725: WT NK cells, stimulated with IL12 + IL18, biol rep 2; Mus musculus; OTHER
250,000 (or 50,000 for input) sorted cells were washed thrice with PBS, resuspended in Buffer 1 (1X eBioscience Perm/Wash Buffer, 1X Roche cOmplete EDTA-free Protease Inhibitor, 0.5 uM Spermidine in H2O) and incubated with STAT4 (Invitrogen 71-4500) antibodies at 1/100 dilution in Antibody Buffer (Buffer 1 + 2uM EDTA) in 96 well V-bottom plate at 4C overnight. Upon antibody incubation, cells were washed twice with Buffer 1 and resuspended in 50ul of Buffer 1 + 1X pA/G-MNase (Cell Signaling #57813) and incubated on ice for 1 hr and washed with Buffer 2 (0.05% w/v Saponin, 1X Roche cOmplete EDTA-free Protease Inhibitor, 0.5 uM Spermidine in 1X PBS) three times. After washing, Calcium Buffer (Buffer 2 + 2uM of CaCl2) was used to resuspend the cells for 30 mins on ice to activate the pA/G-MNase reaction, and equal volume of 2X STOP Buffer (Buffer 2 + 20uM EDTA + 4uM EGTA) was added along with 1pg of Saccharomyces cerevisiae spike-in DNA (Cell Signaling #29987). Samples were incubated for 15 mins at 37C and DNA was isolated and purified using Qiagen MinElute Kit according to manufacturer's protocol Immunoprecipitated DNA was quantified by PicoGreen and the size was evaluated by Agilent BioAnalyzer. When possible, fragments between 100 and 600 bp were size selected using aMPure XP beads (Beckman Coulter catalog # A63882) and Illumina sequencing libraries were prepared using the KAPA HTP Library Preparation Kit (Kapa Biosystems KK8234) according to the manufacturer's instructions with 0.001-0.5ng input DNA and 14 cycles of PCR. Barcoded libraries were run on the NovaSeq X in a PE100 run using 10B reagent kit (200 Cycles) (Illumina). An average of 23 million paired reads were generated per sample.