title: GSM8686028: CTCF_dTAG_rep4; Homo sapiens; OTHER
library_name: GSM8686028
library_construction_protocol: 1.5 M cells were crosslinked with 1% formaldehyde (Thermo Scientific) for 10 min at room temperature, followed by quenching with 125 mM ice-cold glycine (Sigma-Aldrich) for 5 min and 2 washes with ice-cold PBS. Nuclei isolation was performed with Farnham lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40) and followed by nuclear lysis with 0.5% SDS buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% SDS). For CTCF ChIPmentation, chromatin fragmentation was performed by sonication for 7 min with the following settings on the S220 Covaris sonicator: duty factor: 5%, peak incident power: 140 W, cycles per burst: 200). For SSRP1-HA and SMC1A ChIPmentation, chromatin was first digested by a titrated amount of MNase (2.5 kunitz; NEB, M0247S) at 37°C for 10 min and followed by gentle sonication (duty factor: 2%, peak incident power: 105W, cycles per burst: 200) for 1 or 12 min, respectively. The supernatant of the sheared chromatin was mixed with IP buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 150 mM NaCl, 1% Triton-X-100, 1×PIC). Protein A and Protein G dynabeads (Invitrogen) were mixed in a 1:1 ratio and after 3 washes with 0.5% BSA blocking solution (Sigma-Aldrich), the mixture was incubated with 2 μg of primary antibody (HA: Cell Signaling, 3724S; CTCF: Diagenode, C15410210-50; SMC1A: Abcam, ab9262) and 1 μg of spike-in antibody (Active Motif, 61686) for 6 h at 4°C. Sheared chromatin was then added with 200 ng of Drosophila spike-in chromatin to the bead-bound antibodies for 16 h at 4°C. Post-immunoprecipitation washes were performed using buffers with varying salt concentrations at 4°C. Sequencing adapters were added using "on-bead" tagmentation using the Illumina Tagment DNA Enzyme (Illumina, 20034197), after which overnight de-crosslinking was performed at 65°C with proteinase K at a final concentration of 0.2 mg/mL. DNA extraction was performed with 1.8 volumes of Mag-Bind TotalPure NGS beads (Omega Bio-Tek). Library preparation was performed using the NEBNext High-Fidelity 2×PCR Master Mix (NEB, M0541) and two rounds of size selection with 1.2 volumes of Mag-Bind TotalPure NGS beads were performed.