title: GSM5511325: SK-N-BE-2-Ctrl-MYCN; Homo sapiens; ChIP-Seq
library_construction_protocol: 1x10^7 cells were fixed with 1% paraformaldehyde in PBS for 10 minutes and then quenched with 0.125 M glycine for 5 minutes at room temperature. Fixed cells were sequentially lysed with 10 mL lysis buffer 1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100 and 1× protease inhibitors) and 10 mL lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and 1× protease inhibitors). The cells were then resuspended in 300 ml lysis buffer 3 (10 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine and 1× protease inhibitors), and were sheared using the Diagenode Bruptor Plus with the high-power mode for 25 cycles (sonication cycle: 30 seconds ON, 30 seconds OFF). After sonication, 1% Triton X-100 was added to the chromatin and centrifuged at 20,000 g for 10 minutes to remove the insoluble debris. The chromatin was immunoprecipitated with 10 mg individual antibodies and 15 ml pre-blocked Protein A/G beads (Smart-Lifesciences). The beads were washed three times with 1 mL lysis buffer 3, and three times with wash buffer (50 mM HEPES-KOH, pH 7.5, 300 mM LiCl, 1 mM EDTA, 1.0% NP-40 and 0.7% Na-Deoxycholate). The immunoprecipitated DNA was eluted with 200 ml elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1.0% SDS and 200 mg/mL proteinase K) at 55°C for overnight, and purified by phenol-chloroform extraction and ethanol precipitation. Library preparation was done using the NEBNext ultra II DNA library prep kit for Illumina, followed by sequencing on a NovaSeq 6000.