GSM9835086: LEF1  C&R, Sort-purified (CD8+/Ly5.1+/CD44-/CD62L+) TCR-P14 CD8 T cells isolated from spleens replicate #2; Mus musculus; OTHER
After flow sorting, samples were fixed with PFA, quenched with Glycine and transferred to a BSA-coated 96 well plate. Cells were washed and resuspended in buffer (1X eBioscience Perm/Wash Buffer, 1X Roche cOmplete EDTA-free Protease Inhibitor, 0.5 uM Spermidine, + 2uM EDTA in H2O) containing anti-LEF1, anti-TCF1, anti-H3K4me3 (positive control) or no antibody (negative input control) overnight. The following day, cells were resuspended in buffer (1X eBioscience Perm/Wash Buffer, 1X Roche cOmplete EDTA-free Protease Inhibitor, 0.5 uM Spermidine in H2O)  containing pAG-MNase (CELL SIGNALING) for 1 hour before wash and incubation in calcium-containing buffer (0.05% w/v Saponin, 1X Roche Complete EDTA-free Protease Inhibitor, 0.5 uM Spermidine in 1X PBS + CaCl2 2uM) for 30 minutes. After incubation, STOP buffer (0.05% w/v Saponin, 1X Roche cOmplete EDTA-free Protease Inhibitor, 0.5 uM Spermidine in 1X PBS+ 20uM EDTA + 4uM EGTA) was added to the samples, supernatant was collected, and DNA was isolated with the MiniElute Kit. DNA fragments were digested with Proteinase K (Thermo Fisher, cat. EO0491) under addition of 0.1% SDS for 4h at 65°C. Library prep was conducted on the product using a KAPA HyperPrep Kit (Kapa Biosystems, cat. KK8504). The final product was cleaned by AMPure XP beads (Beckman Coulter catalog no. A63882).
GEO
GSM9835086_r1