GSM8195739: BLaER, SMC1A_ChIP_24h_rep2; Homo sapiens; ChIP-Seq
Aliquots of 1.5 M cells were crosslinked in a single fixation reaction with 1% formaldehyde for 10 min at room temperature, quenched with ice-cold glycine at a final concentration of 125 mM, and washed 2 times with ice-cold PBS. Fixed cells were first gently lysed with Farnham lysis buffer (5 mM PIPES pH 8, 85 mM KCl, 0.5% NP-40) to isolate nuclei, which was followed by nuclear lysis with 0.5% SDS buffer (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5% SDS). Then, chromatin was fragmented, using an optimized procedure for each target. In the CTCF ChIPmentation experiments, chromatin was fragmented by sonication (time: 7 min, duty factor: 5 %, peak incident power: 140 W, cycles per burst: 200). In MED26 and SMC1A ChIPmentation experiments, chromatin was first fragmented by a titrated amount of MNase (2.5 Kunitz) and then gently sonicated (time: 1 min, duty factor: 2 %, peak incident power: 105 W, cycles per burst: 200). To perform immunoprecipitation, a mixture of Protein A (10008D, Invitrogen) and Protein G Dynabeads (10003D, Invitrogen) in 1:1 ratio was blocked with 0.5 % BSA, washed and pre-incubated with 2 µg of primary antibody (CTCF: Diagenode, C15410210-50; SMC1A: Abcam, ab9262; MED26: Bethyl Laboratories, A302-370) and 1 µg of spike-in antibody (Biozol, 61686) for 6 h, at 4 °C. Sheared chromatin was diluted in IP buffer, and added along with 50 ng Drosophila spike-in chromatin to the bead-bound antibodies. The samples were incubated overnight on a rotator at 4 °C, and washed with stringent buffers the next morning. Sequencing adapters were added to the bead-bound DNA by Tagmentation with the Illumina Tagment DNA Enzyme and Buffer Kit (Illumina, 20034197). After washes, the bead-bound samples were de-crosslinked overnight at 65 °C in the presence of proteinase K (0.2 mg/mL, final) and cleaned with 1.8x volume of Mag-Bind TotalPure NGS beads (Omega Bio-Tek, M1378-01). The tagmented DNA was used for library preparation with the NEBNext High-Fidelity 2X PCR Master Mix (NEB, M0541).