GSM5225988: FLAG_PFDN5_input_rep1; Homo sapiens; ChIP-Seq
Human chromatin immunoprecipitation was carried out by fixation (10 minutes, 1 % formaldehyde, 37 ºC) and quenching (5 minutes, 2 % glycine), cells were lysed in lysis buffer (50 mM Tris-HCl, 10 mM EDTA, 1% SDS, pH 8.1 and protease inhibitors). Samples were then sonicated (Bioruptor, Diagenode) with cycles of 30 seconds for 15 minutes and the extracts of 300 bp obtained were cleared by centrifugation. Sonicated chromatin were incubated with 50 µl of magnetic dynabeads protein A o G (Life technology) coupled to anti-FLAG (Sigma, F3165, ChIP) antibody-.  Immunoprecipitated material was washed with three different wash buffers (wash buffer 1: 0.1 % (w/v) SDS; 1 % (v/v) Triton-X100; 2 mM EDTA; 20 mM Tris-HCl pH 8; 150 mM NaCl. Wash buffer 2: 0.1 % (w/v) SDS; 1 % (v/v) Triton-X100; 2 mM EDTA; 20 mM Tris-HCl pH 8; 500 mM NaCl. Wash buffer 3: 0.25 M LiCl; 1 % (v/v) NP-40; 1 % (w/v) Sodium deoxicholate; 1 mM EDTA; 10 mM Tris-HCl pH 8) and TE (10 mM Tris-HCl pH 8; 1 mM EDTA). DNA was eluted using elution buffer (1 % (w/v) SDS; 0.1 M NaHCO3) and, treated with 5 µl proteinase K (20 mg/ml) for 1 hour at 37 ºC. The resultant DNA was fenol-chloroform extracted, and precipitated.