title: GSM6208878: ChIPSeq_CTCF_R2_HOT_plus; Mus musculus; ChIP-Seq
library_construction_protocol: iMEF cells were treated with HOT or vehicle for 7 days and cross-linked with 1% formaldehyde in cell culture medium on a shaking platform for 10 min at room temperature (RT). The cross-linking reaction was quenched by adding glycine to 125 mM and incubation for another 5 min at RT. Adherent cells were washed with PBS twice and collected in PBS by scraping the cells from the dish. Cells were centrifuged at 380 x g for 5 min at 4°C and resuspended in 1 ml Lysis Buffer B (10 mM Hepes pH 6.5, 10 mM EDTA, 0.5 M EGTA, 0.25% (v/v) Triton X-100) per  107 cells and centrifuged. To the pellet 1 ml Lysis Buffer C (10 mM Hepes pH 6.5, 10 mM EDTA, 0.5 M EGTA, 200 mM NaCl) was added per 107 cells, followed by an incubation on ice for 10 min. After a further centrifugation step, disruption of the pelleted nuclei was performed in 100 μl lysis buffer D (50 mM Tris pH 8, 10 mM EDTA, 1% (w/v) SDS, protease inhibitor cocktail (1xPIC, Merck, P8340) per 107 MEF cells for 30 min at 15°C. 300 μl aliquots were sonicated for a minimum of 5 cycles using a Bioruptor Pico (Diagenode) with 30-second on-off-cycles to achieve DNA fragment lengths with a majority around 300 bp for  ChIP-seq, respectively. The size distribution of the resulting DNA fragments was assessed by agarose gel electrophoresis. To remove cell debris, lysates were centrifuged for 10 min at 10,000 x g (15°C), the supernatants pooled, and DNA concentrations were measured using a NanoDrop 1000 device (Peqlab). ChIP assays were performed using the OneDay ChIP Kit from Diagenode (C01010080) according to the manufacturer's instructions. The sheared chromatin (100 µg) was diluted 4.7-fold with 1 x ChIP buffer, setting aside 1/10 of the diluted chromatin for the input control. The diluted chromatin was supplemented with 5 µg of CTCF-specific antibody. This immunoselection was done at 4°C in a rotator overnight. To capture the antibody-chromatin-complexes, 42 µl of Protein A beads per sample were washed with 1x ChIP buffer. After the washing, the supernatant of the beads were discarded and the pre-incubated antibody-chromatin-complexes were added to the beads. The IP was done for 90 min at 4°C. Subsequently, the chromatin-antibody-bead complexes were washed three times with 1 x ChIP. The diluted input control chromatin was precipitated by the addition of 5 volumes 100% ethanol and centrifuged at 16,000 x g, 4°C for 10 min. The dried pellets were resuspended in RNase-free. The DNA of all samples (including input) were reverse crosslinked and further purified with the QIAquick PCR purification Kit (28106). The libraries were prepared using the Next Ultra II kit (New England BioLabs) and the sequencing was performed as single reads for 75 cycles with a NextSeq 500/550 High Output kit v2 (Illumina) according to the manufacturer's recommendations.