title: GSM5225845: Pt-ttpy_rep1; Homo sapiens; ChIP-Seq
library_construction_protocol: After stopping the fixation by glycine (0.125M) the cells are recovered by scraping, centrifuged and lysed with the lysis buffer (5mM PIPES pH8, 85mM KCl, 0.5% NP40, 1X inhibitor cocktail) using piston B (dounce homogenizer) 10 times in ice. The lysed cells are then aliquoted (approximately 2-4 million per tube) the nucleus was then lysed by the buffer (5mM Tris-HCl pH8, 10mM EDTA, 1% SDS, 1X inhibitor cocktail) and sonicated to obtain fragments of size 200-700bp. The sonicated chromatin was then incubated with ChIP Adembeads protein A / G (Sigma) which have already blocked for 15 minutes with 225 μl of “blocking buffer” and incubated with 1-3 μg of anti γH2AX antibody (anti -rabbit ab2893) in IP buffer (0.01% SDS, 1.1% triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl pH8, 16.7mM NaCl) The beads were then washed with several washing buffers which contain increasing concentrations of NaCl, then the magnetic particles are suspended in 300 μl of elution buffer (0.1M NaHCO3, 1% SDS) and proteinase K. DNA was recovered and purified using phenol / chloroform , and its concentration assayed with Nano-drop and qubit. DNA quality was assayed by Agilent 2100 expert high sensitivity DNA assay and the library prepared and high-throughput sequencing done on HiSeq2500 (Rapid Flow cell,50 bases Single Read, Illumina TruSeq Read)