GSM8625676: IgG_SV_HFD_1; Mus musculus; ChIP-Seq
Frozen tissue was Dounce homogenized and fixed with 20 strokes using the loose pestle A followed by 20 strokes with the tight pestle B in Cell Lysis Buffer (5 mM PIPES pH 8, 85 mM KCl, 1% Igepal, 1× protease inhibitor) containing 0.5% formaldehyde. The mixture was rotated for 8 minutes at room temperature. The cross-linking reaction was quenched by adding 150 mM glycine for 5 minutes at room temperature. Samples were washed twice with ice-cold Cell Lysis Buffer supplemented with 1× protease inhibitors. The pellet was suspended in 300 µl of SDS Lysis Buffer (50 mM Tris-Cl, 0.5% SDS, 10 mM EDTA, 1× protease inhibitor) and incubated on ice for 1 hour. Chromatin was sheared using a Bioruptor Plus sonicator (Diagenode) for 35 cycles at a high setting (30s ON, 30s OFF per cycle), allowing the sonicator to cool down for 5 minutes after every 10 cycles. Triton-X-100 was added to a final concentration of 1% along with 1× protease inhibitors. Samples were centrifuged at 13,000 rpm for 20 minutes, and the supernatant was collected for immunoprecipitation. 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.