GSM8606392: B cell, Input; Mus musculus; OTHER
Following stimulation, cell were washed thrice with PBS, and resuspended in Antibody Buffer (1X eBioscience Perm/Wash Buffer, 1X Roche cOmplete EDTA-free Protease Inhibitor, 0.5 μM Spermidine, 2 μM EDTA in H2O). Cells were then incubated overnight with anti-STAT3 Mouse mAb (124H6, CellSignaling, Cat #9139, dilution 1/100) in Antibody Buffer in a 96 well V-bottom plate at 4°C. Cells were then washed twice with Buffer 1 (1X eBioscience Perm/Wash Buffer, 1X Roche cOmplete EDTA-free Protease Inhibitor, 0.5 μM Spermidine in H2O) and resuspended in 50ul of Buffer 1 + 1X pA/G-MNase (Cell Signaling, cat. 57813) and incubated on ice for 1 hour and washed thrice with Buffer 2 (0.05% w/v Saponin, 1X Roche cOmplete EDTA-free Protease Inhibitor, 0.5 μM Spermidine in 1X PBS). After washing, cells were resuspended in calcium Buffer (Buffer 2 + 2 μM CaCl2) for 30 mins on ice to activate the pA/G-MNase reaction, and equal volume of 2X STOP Buffer (Buffer 2 + 20 μM EDTA + 4 μM EGTA) was added along with 1 pg of Saccharomyces cerevisiae spike-in DNA (Cell Signaling, cat. 29987). Samples were incubated for 15 mins at 37°C and DNA was isolated and purified using Qiagen MinElute Kit according to manufacturer's protocol and subjected to library amplification. DNA was quantified by PicoGreen and the size was evaluated by Agilent BioAnalyzer. Illumina sequencing libraries were prepared using the KAPA HTP Library Preparation Kit (KAPA Biosystems #KK8234) according to the manufacturer's instructors with <0.001-3.8 ng input DNA and 14 cycles of PCR.