GSM9893306: P2S5p; Homo sapiens; ChIP-Seq
MCF10A cells were cross-linked 10 min at RT by directly adding formaldehyde to the culture medium to a final concentration of 1%. Cross-linking was stopped by addition of glycine to a final concentration of 125 mM. Cells were washed twice with ice-cold PBS and lysed in SDS buffer: 100 mM NaCl, 50 mM Tris–HCl (pH 8), 5 mM EDTA (pH 8), 0.5% SDS and protease inhibitors (10 min, RT). Chromatin lysates were diluted in Triton Dilution Buffer(100 mM Tris–HCl at pH 8, 100 mM NaCl, 5 mM EDTA at pH 8, 5% Triton X-100, and protease inhibitors) by adding ½ V. Sonication was performed with Branson Digital Sonifier 250 to obtain fragments in the range of 500-250 bp. Antibody incubations were performed overnight with the following: anti-Pol II-pS5 (ab5131 Abcam, Cambridge, UK), anti-Pol II-pS2 (A300-654A, Bethyl Laboratories, Montgomery TX, USA), anti-53bp1 (ab36823, Abcam, Cambridge, UK) Libraries were prepared with NEBNext Ultra II DNA