title: GSM4776736: IP RNAPII Ctrl R2; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were cross-linked (1% formaldehyde), harvested, lysed and sonicated (Covaris S220 ultrasonicator). Chromatin (40 µg per antibody) was diluted and precleared on Protein A/G Dynabeads for 30 min at 4°C under rotation. Beads were removed and 1% of chromatin was taken apart as Input material. Antibodies were added to chromatin and incubated overnight at 4°C under constant mixing, before being bound to Protein A/G Dynabeads for 5h.   Immunoprecipitated material was washed once with Low Salt buffer, once with High Salt Buffer, once with LiCl Buffer and twice with Tris/EDTA buffer, before being eluted and reverse crossed-linked  overnight at 65°C on Thermomixer (Eppendorf) in the presence of 200 mM NaCl and 20 µg proteinase K (Roche). DNA was then purified by phenol extraction and ethanol precipitation, and quantified for library preparation. 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.