Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: ChIP-seq experiments were carried out as follows. Briefly, 2x10^6 K562 cells were pelleted at 2000 g for 5 minutes at 4C and then resuspended in 1x PBS buffer; 37% formaldehyde solution (Sigma F8775) was added at a final concentration of 1%. Crosslinking was carried out at room temperature for 15 minutes, and then the reaction was quenched by adding 2.5 M Glycine solution at a final concentration of 0.25 M. Crosslinked cells were then pelleted at 2000 g for 5 minutes at 4C, washed with cold 1x PBS buffer, and stored at -80C.. CTCF ChIP was performed using a polyclonal anti-CTCF antibody (Millipore, 07-729). For each reaction, 100 uL of Protein A Dynabeads (Thermo Fisher 10001D) were washed 3 times with a 5 mg/mL BSA (Sigma A9418) solution. Beads were then resuspended in 1 mL BSA solution and 4 uL of CTCF antibody were added. Coupling of antibodies to beads was carried out overnight on a rotator at 4C. Beads were again washed 3 times with BSA solution, resuspended in 100 uL of BSA solution, mixed with 900 uL sonicated chromatin and incubated overnight on a rotator at 4C. Chromatin was sonicated using a tip sonicator (Misonix) after cells were lysed with Farnham Lysis Buffer (5 mM HEPES pH 8.0, 85 mM KCl, 0.5% IGEPAL, Roche Protease Inhibitor Cocktail), and nuclei were resuspended in RIPA buffer (1x PBS, 1% IGEPAL, 0.5% Sodium Deoxycholate, 0.1% SDS, Roche Protease Inhibitor Cocktail). The sonicated material was centrifuged at 14,000 rpm at 4C for 15 minutes to remove cellular debris, and a portion of the supernatant was saved as input. After incubation with chromatin, beads were washed 5 times with LiCl buffer (10 mM Tris-HCl pH 7.5, 500 mM LiCl, 1% NP-40/IGEPAL, 0.5% Sodium Deoxycholate) by incubating for 10 minutes at 4C on a rotator and then rinsed once with 1x TE buffer. Beads were then resuspended in 200 uL IP Elution Buffer (1% SDS, 0.1 M NaHCO_3) and incubated at 65C in a Thermomixer (Eppendorf) with interval mixing to dissociate antibodies from chromatin. Beads were separated from chromatin by centrifugation, Proteinase K was added to the supernatant and crosslinks were reversed at 65C for \sim16 hours. Input samples (100 uL) were mixed with an equal volume of IP Elution Buffer, Proteinase K was added and cross-links were reversed together with the ChIP samples. DNA was purified by phenol-chloroform-isoamyl extraction followed by MinElute column (Qiagen) clean up. DNA concentration was measured using QuBIT, and libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S). Libraries were sequenced on a NextSeq (Illumina) in a 2x75 bp format.