GSM5327302: CTCF CT rep1; Homo sapiens; ChIP-Seq
Between 10-20 millions cells  were cross-linked with 1% formaldehyde in fixation solution directly in culture dishes and incubated for 5 minutes at RT and then 40 minutes at 4°C. Cross linking reaction was stopped by addition of 0.125M Glycine. For ChIP-seq, we used the iDeal ChIP-seq kit for Transcription Factors and iDeal ChIP-seq kit for Histones (Diagenode). Chromatin extraction, immunoprecipitations and DNA purifications is performed as recommended by the manufacturer ChIP samples were purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified with the Qubit (Invitrogen). ChIP-seq libraries were prepared from 10 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (C05010014, Diagenode s.a., Seraing, Belgium), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5 prime ends were ligated to the 5 prime end of the genomic DNA, leaving a nick at the 3 prime end. The adaptors cannot ligate to each other and do not have single-strand tails, avoiding non-specific background. In the final step, the 3 prime ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (7  cycles). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter) to remove unincorporated primers and other reagents.