title: GSM5905310: CTCF_SV_HFD_2 [F_SV_113_5]; Mus musculus; ChIP-Seq
library_construction_protocol: Frozen tissues were Dounce homogenized with loose pestle A in 1× PBS with 1× protease inhibitors (cOmplete, Roche). Tissue suspension was centrifuged at 2000 rpm for 5 min and the pellet was resuspended in 1 ml PBS containing 1 % formaldehyde for 10 min and quenched with glycine 150 mmol/L for 5 min at room temperature. Samples were washed twice with ice-cold 1× PBS with 1× protease inhibitors. Cells were lysed by pelleting and diluting twice in ice-cold SDS lysis buffer (50 mM Tris/HCl, 0.5% SDS, and 10 mM EDTA, 1 × protease inhibitor) and incubating for 10 min at 4°C with gentle mixing. The cell lysate was then Dounce homogenized with tight pestle B in 1 ml ice-cold of SDS lysis buffer and the homogenate was filtered through a 100 µm cell strainer. Samples were split into 3 × 300 µl aliquots in 1.5 sonication tubes and incubated on ice for 10 min. Chromatin was sheared with Bioruptor Plus sonicator (Diagenode) for 35 cycles at high setting. Once cycle was 30s ON, 30s OFF and after each 10 cycles the sonicator was allowed to cool down for 5 min. To neutralize SDS, Triton-X-100 was added to final concentration of 1% along with 50× protease inhibitors (final 1×). All the aliquots were combined and centrifuged at 13,000 rpm for 20 min. Supernatant with sheared chromatin was collected. ChIP and library preparation were performed using the recently described ChIPmentation protocol (Gustafsson C et al. BMC Genomics. 2019;20(1):59) with minor modifications. For ChIP, 2 µg of rabbit polyclonal anti-CTCF antibody, 5µl of rabbit monoclonal anti-TCF4/TCF7L2 antibody or 2 µg of Anti-Histone H3 (acetyl K27) antibody was added to 50 µl Protein G-coupled Dynabeads (Thermo Fisher Scientific) in 1 × PBS with 0.5% bovine serum albumin (BSA) and rotated at 40 rpm for 4 h at 4°C. Dynabeads were washed three times with PBS with 0.5% BSA and then mixed with 190 µl chromatin samples in 1.5 ml tubes. The samples were rotated at 40 rpm overnight at 4°C. Chromatin was washed with 150 µl of low-salt buffer (50 mM Tris/HCl, 150 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, and 1 mM EDTA), high-salt buffer (50 mM Tris/HCl, 500 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, and 1 mM EDTA) and LiCl buffer (10 mM Tris/HCl, 250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, and 1 mM EDTA), followed by two washes with TE buffer (10 mM Tris/HCl and 1 mM EDTA) and two washes with ice-cold Tris/HCl pH 8. Chromatin was resuspended in 30 µl of 2 × TD buffer and 1 µl of transposase (Nextera, Illumina) for tagmentation. Samples were incubated at 37°C for 10 min followed by two washes with low-salt buffer. Bead bound tagmented chromatin was diluted in 23 µl of water and 25 µl of 2 × Ultra II Q5 Master Mix (New England BioLabs, M0544S) and 1 µl of both amplification primers (Buenrostro et al, 2013; 0.125 µM final concentration) were added. The libraries were incubated as follows: 72 °C 5 min (adapter extension); 95 °C 5 min (reverse cross-linking); followed by 11 cycles of 98 °C 10s, 63 °C 30s and 72 °C 3 min. After PCR amplification, double-sided purification was performed using SPRIselect beads (Beckman Coulter). To prepare input controls, 2 μl of 50 mM MgCl2 was added to 10 μl sonicated lysate to neutralize the EDTA in the SDS lysis buffer. Tagmentation and amplification was done as described above.