GSM6788437: NPC_CTCF_H3K27ac_Input; Homo sapiens; ChIP-Seq
We fixed the cells for all downstream ChIP-seq assays as previously described (Beagan et al., 2017; Beagan et al., 2016; Beagan et al., 2020; Phillips-Cremins et al., 2013). Briefly, we fixed 8-10 million cells by adding 1 % (v/v) formaldehyde in DMEM/F-12 (Thermo Fisher, 11320033) for 10 min at room temperature. The complete fixation media was 50 mM HEPES-KOH (pH 7.5), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 11 % formaldehyde. We quenched the fixation process by adding 125 mM glycine for 5 min at room temperature, following by 15 min at 4 °C. We washed the cross-linked cells in pre-chilled PBS before flash freezing and stored the pellets at −80 °C. We then lysed in pre-chilled cell lysis buffer (10 mM Tris pH 8.0, 10 mM NaCl, 0.2 % (v/v) NP-40, 1 mM PMSF, 0.2 % (v/v) Protease inhibitor cocktail (Sigma, P8340)) and incubated on ice for 10 min. We homogenized cells using dounce homogenizer with pestle A (30 times). We centrifuged lysates at 2,500 g for 5 min at 4 °C to pellet nuclei. We solubilized nuclei in 500 μl nuclear lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1 % (w/v) SDS, 1 mM PMSF, 0.2 % (v/v) Protease inhibitor cocktail) on ice for 20 min before mixing with 300 μl IP dilution buffer (20 mM Tris pH 8.0, 2 mM EDTA, 150 mM NaCl, 1 % (v/v) Triton X-100, 0.01 % (w/v) SDS, 1 mM PMSF, 0.2 % Protease inhibitor cocktail). Next, we sheared chromatins to 200-600 bp DNA fragment size using Qsonica Q800R3 (Qsonica Sonicators, CT) with parameters of 100 % amplitude and 30 sec on / 30 sec off pulses. After sonication we centrifuged lysates at 16,000 g and added pre-clearing solution (50 μg of IgG (Sigma, I8140), 175 μl of Protein A Agarose (Thermo Fisher, 15918014) and 175 μl of Protein G Agarose (Thermo Fisher, 15920010) in 3.7 ml of pre-chilled IP dilution buffer and 0.5 ml of nuclear lysis buffer) to each supernatant. We incubated the samples by rotating at 10 rpm for 2 hrs at 4 °C. Subsequently, we performed target protein-associated chromatin immunoprecipitation from supernatant using antibody-bound beads by rotating the samples at 10 rpm overnight at 4 °C. Next day, we washed pull down beads once in IP wash buffer 1 (20 mM Tris pH 8.0, 2 mM EDTA, 50 mM NaCl, 1 % (v/v) TritonX-100, 0.1 % (w/v) SDS), twice in high salt buffer (20 mM Tris pH 8.0, 2 mM EDTA, 500 mM NaCl, 1 % (v/v) TritonX-100, 0.01 % (w/v) SDS), once in IP wash buffer 2 (10 mM Tris pH 8.0, 1 mM EDTA, 250 mM lithium chloride, 1 % (v/v) NP-40, 1 % (w/v/) sodium deoxycholate), twice in 1 x TE at 4 °C. We eluted chromatins from beads in elution buffer (1 % (w/v) SDS, 100 mM sodium bicarbonate), and degraded RNA by adding RNaseA (Roche, 10109169001) at 65 °C for 1 hr. To remove residual proteins and reverse crosslink DNA, we added 2.4 U of Proteinase K (NEB, P8107S) to eluent and incubated at 65°C overnight. We purified ChIP DNA using phenol chloroform extraction and ethanol precipitation methods. We stored ChIP DNA at -20 °C until we performed library preparation. We used 5 ng of purified ChIP-seq DNA for downstream library preparation. We prepared all sequencing libraries using the NEBNext Ultra II Library Prep Kit (NEB, E7645S) following the manufacturer's protocol. We first end repaired, and dA-tailed DNA from ChIP experiments and ligated with Illumina adaptors using NEBNext Ultra II DNA Library Prep Kit for Illumina according to the manufacturer's protocol with minor modifications. DNA fragments <1 kb size were size-selected and amplified in subsequent steps using 7-8 PCR cycles. After additional purification using AgenCourt Ampure XP beads (Beckman Coulter, A63881) according to the manufacturer's instruction, we assessed quality of the individual libraries using Agilent Bioanalyzer High Sensitivity DNA Analysis Kits (Agilent, 5067-4626) and quantified using Kapa Library Quantification Kit (KAPA biosysytem, KK4835) prior to library pooling sequencing on Illumina NextSeq 500. We sequenced ChIP-seq libraries with reading length of 75 bp single-ended.