GSM4909449: NSC_Rep1_3xBG4_rep3; Homo sapiens; ChIP-Seq
G4-ChIP-seq was performed as previously described (Hansel-Hertsch et al 2018) with the following modifications. 30 million cells were dissociated with Accutase and cross-linked in serum-free media containing 1% (v/v) methanol-free formaldehyde for 10 min and then quenched with 0.12M glycine for 10 min at room temperature. Cross-linked cells were washed with PBS by centrifugation and flash frozen in liquid nitrogen and stored at -80 °C. Chromatin was isolated using the Lysis and Hypotonic Buffer for Sonication kit (Chromatrap). Each lysed nuclei suspension was sonicated for 25-40 cycles (30 sec on/ 60 sec on, high setting) using a water-cooled bath sonicator until an average DNA size of 100-500 bp was reached.  For ChIP, 7.5 µL of 150 ng/mL chromatin was incubated in Blocking Buffer (25 mM HEPES pH 7.5, 10.5mM NaCl, 110m KCl, 1mM MgCl2 and 1% (w/v) bovine serum albumin) supplemented with 3 µL 1mg/ml RNaseA for 30 min at 800 rpm on a thermoshaker at 16 °C. Next 6 µL of 4.8µM BG4 anti-G4 antibody (generated via autoinduction as previously described in Biffi et al 2014) was added and the reaction incubated for 1 h at 16 °C with mixing at 1,400 rpm. After pre-blocking anti-flag M2 magnetic beads (Merck) with Blocking Buffer for 1 hour at 4°C, 150 µL of this bead slurry solution was then added to the CHIP reaction and incubated for 1 h at 16 °C with mixing at 1,400 rpm. Beads containing antibody bound chromatin were magnetically captured, supernatant discarded via vacuum aspiration and washed four times with wash buffer (10 mM Tris pH 7.4, 100 mM KCl, 0.1% (v/v) Tween 20) at 4°C by manual agitation.  Beads were then subjected to two subsequent washes at 37 °C in a thermoshaker at 1,400 rpm for 10 min. The supernatant was then removed, beads resuspended in 150 µL of elution buffer (1 x TE buffer, 3 µL proteinase K) and incubated at 65 °C  with 1,400 rpm shaking for 3 h to digest protein.  DNA from ChIP and input samples was then purified using MinElute Reaction Clean up Kit. For library preparation, samples were quantified by Qubit dsDNA HS assay and 10ng of purified ChIP or input DNA was tagmentated (2.5 µL Tn5 (Illumina) in tagmentation buffer (Illumina)) in total reaction volume of 40 µL at 800 rpm shaking for 20 min at 37 °C. After purification by MinElute Reaction Clean up Kit, libraries were generated by mixing 20 µL tagmentated DNA, 2.5 µL each of Nextera Index i7 and Nextera Index i5 (Nextera index primer kit, Illumina) with 25 µL NEB Next High Fidelity 2 x PCR master mix (NEB) in a PCR reaction using the following conditions: 72 °C for 5 min, 98 °C for 30 sec followed by six cycles of 98 °C for 10 sec, 63 °C for 30 sec and 72 °C for 1 min. Libraries were purified by MinElute reaction clean-up kit and library quality and quantity were assessed by Bioanalyser and Qubit, respectively. Samples were sequences on a NextSeq platform (single-end, 75 bp reads).