title: GSM2257818: CTCF_E3h_ChIPseq; Homo sapiens; ChIP-Seq
library_construction_protocol: For each ChIP-seq, 2 plates 70-80% confluent (around total 20 millions MCF7 cells) were lysed in SDS buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0)) and sonicated for 20 cycles 30 sec ON 30 sec OFF in Bioruptor sonicator (Diagenode). Subsequently, chromatin was diluted in dilution buffer (10 mM Tris-HCl pH 8.0, 0.5  mM EGTA pH 8.0, 1% Triton-X-100, 140 mM NaCl) and incubated over night with 10 ug of antibody coupled with magnetic beads. Afterwards, beads were washed 7 times with RIPA buffer (50mM HEPES pH7.6, 1mM EDTA pH8, 0.7% Na-deoxycholate, 1% NP-40, 0.5 M LiCl2) and reverse cross-linked at 65 degrees overnight in Elution buffer (50 mM Tris-HCl pH8, 1 mM EDTA pH8, 1% SDS). RNA was digested at 37 degrees in RNAse A (Ambion), and proteins were digested with proteinase K (Life Technologies) at 56 degrees. DNA was isolated by Phenol-chloroform extraction followed by Ethanol precipitation. Library preparation for sequencing was done following the instructions of TruSeq DNA sample preparation kit from Illumina.