Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Cells were on-plate crosslinked with 1% of formaldehyde (Sigma F1635-500ml) during precisely 8 minutes, then 0.125 M of Glycine (Axonlab A1067.5000) was added to terminate the crosslinking reaction. Cells were washed twice with cold PBS 1X and lyzed for 10 min on ice in 0.5% NP40 lysis buffer (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP40, supplemented with one tablet of complete EDTA-free Protease Inhibitor Cocktail (Roche 04693132001) per 50 mL; 950 uL per 10 million of cells). The nuclei were recovered by high-speed centrifugation (5 minutes at 3200 x g, at 4 C), resuspended in nuclei lysis buffer (NLB, 50 mM Tris-HCl pH 8.1, 10 mM EDTA pH 8.0, 1% SDS, supplemented with one tablet of complete EDTA-free Protease Inhibitor Cocktail) and incubated for 20 minutes at 4°C. Chromatin was sonicated using a Bioruptor Pico (Diagenode) to obtain fragments of around 200 base pairs. Sonicated chromatin was clarified by centrifugation and subsequently 1:2 diluted in 2X IP buffer (33.4 mM Tris pH 8.1, 167 mM NaCl, 167 mM LiCl, 2.4 mM EDTA pH 8.0, 2.2% Triton X-100, 0.02% SDS, supplemented with one tablet of complete EDTA-free Protease Inhibitor Cocktail) before being snap frozen in liquid nitrogen. A 50 uL aliquot was kept to analyze the chromatin quality and concentration. The frozen sonicated chromatin was stored at - 80°C. The day prior to the chromatin immunoprecipitation, protein G agarose beads (Roche 1243233) were washed with NLB:IP buffer (a mix of equal amounts of NLB and 2X IP buffer) and further incubated overnight under rotation at 4°C) in NLB:IP buffer supplemented with 100 ug/mL of Bovine Serum Albumine (BSA, Sigma A8022-100). The sonicated chromatin was thawed. A 60 uL aliquot was kept for the input sample, while, for each IP, 9 ug of chromatin (in a total volume of 1200 uL of NLB:IP buffer) was overnight incubated under rotation at 4°C with 2 ug of anti-THAP11 antibody. Samples were further incubated under rotation at room temperature with 60 uL of the above washed and BSA-blocked protein G agarose beads. Immunoprecipitated samples were washed twice with IPWB1 buffer (IP wash buffer 1: 20 mM Tris pH 8.1, 50 mM NaCl, 2 mM EDTA pH 8.0, 1% Triton X-100 and 0.1% SDS). Each IP was performed 5 times in parallel as described and subsequently pooled to form a single IP sample at this step, after the washes with IPWB1 buffer. Pooled IP samples were then washed once with IPWB2 bffuer (IP wash buffer 2: 10 mM Tris pH 8.1, 250 mM LiCl, 1 mM EDTA pH 8.0, 1% NP40 and 1% Na-deoxycholate) and finally twice with TE buffer (10 mM Tris pH 8.1, 1 mM EDTA pH 8.0). Two elutions with IPEB buffer (elution buffer: 100 mM NaHCO3, 1% SDS) were sequentially done (5 minutes at 37°C under agitation) and pooled. The input sample was thawed and supplemented with 190 uL of IPEB buffer. Eluates (IP) and input samples were decrosslinked by overnight incubation at 65°C in presence of 20 ug/mL of RNAse A (DNAse-free RNAse A, Roche 1119915) and 300 mM of NaCl. The samples were further incubated during 90 minutes at 45°C with 350 ug/mL of proteinase K (Promega V3021). Samples were finally purified using the Nucleospin Gel and PCR clean-up kit (Macherey-Nagel 740609) using the NTB buffer for SDS-containing samples. Samples were eluted with 50 uL of the pre-warmed (72°C) buffer NE (from the kit), and subsequently re-eluted using the previous eluate. DNA yield was quantified using a Qubit spectrophotometer. For each IP sample, two separate libraries were prepared and sequenced, using the same immunoprecipitated-DNA material. For this, 5 ng of purified DNA was used to prepare paired-end sequencing libraries using the MicroPlex Library Preparation kit (Diagenode C05010014) following the manufacturer instructions. Here, 8 PCR cycles were done for DNA amplication and the DNA fragments were not size selected. Then, libraries were purified using AMPure XP magnetic beads (Diagenode) and entrusted to the UNIL Genomic Technology Facility (GTF) for 100-nucleotide paired-end high-throughput sequencing (Illumina, HiSeq 2100) with 3 samples per line (multiplexing).