GSM4002958: anti-H3K27me3H3K27me3_1_NIH3T3_CUT&RUN; Mus musculus; OTHER
Briefly, approximately 200,000 NIH3T3 cells were washed in wash buffer (20 mM HEPES, pH7.5, 150 mM NaCl, 0.5 mM Spermidine and complete protease inhibitor (EDTA-free, Roche), captured with Concanavalin A beads (Polysciences, Warrington, PA) and incubated with primary antibodies overnight at 4°C. After washing with Dig-wash buffer (20 mM HEPES, pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.08% Digitonin and protease inhibitors), cells were resuspended in 50 μL Digwash buffer and 2.5 μL of protein A-MNase (1:10 diluted, batch 6 from Steve Henikoff) and incubated at room temperature for 10 minutes. Cell pellets were washed again and placed in a 0°C metal block, and 2 mM of CaCl2 was added and incubated for 45 minutes. MNase reaction was terminated by the addition of 2XSTOP buffer and incubated at 37°C 10 minutes. Samples were then digested by proteinase K at 70°C for 10 minutes and DNA was extracted by ethanol precipitation. Library were prepared using KAPA Hyper Prep Kit (KAPA) and custom Y-shaped TruSeq adapters according to the manufacturer's instructions. The antibodies used were anti-Yap (Novus, NB110-58358), anti-H3K27ac (Abcam, ab4729), anti-H3K27me3 (Cell Signaling, 9733S), anti H3K4me3 (Diagenode, C15410003), anti-CTCF (Diagenode, C15410210), anti-GABPA (ProteinTech, 215420-1), Gabpb1 (ProteinTech, 12597-1).