GSM7230513
For ChIP-seq, cells were fixed for ten minutes with direct addition of 1% formaldehyde (ThermoFisher, Waltham, MA). The fixation was stopped with 0.125 mM glycine (Sigma-Aldrich, St. Louis, MO) for five minutes, followed by wash and collection in cold PBS and subsequent lysis (1% SDS, 10 uM EDTA, 50 uM TrisCl pH 8.0, 5 mM sodium butyrate, cOmpleteTM protease inhibitor cocktail tablets) (Sigma-Aldrich, St. Louis, MO). Chromatin was sheared to 300-500 kb using the Bioruptor (Diagenode, Denville, NJ). Lysates were mixed 1:4 with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 uM EDTA, 16.7 uM Tris-Cl pH 8.0, 167 uM NaCl, 5mM sodium butyrate, protease inhibitor cocktail tablet). Immunoprecipitation was completed for four hours using antibodies directed against H3K27ac, MYC, MYCL1, and MYCN with M280 Sheep Anti-Rabbit IgG Dynabeads (ThermoFisher, 11203D). ChIPs were washed 1x with TE and 3x with Wash Buffer (100 mM Tris-Cl pH 8.0, 500 mM LiCl, 15 Igepal, 1% deoxycholic acid) supplemented with PMSF. Beads were resuspended in Elution Buffer (20 mM NaHCO3, 1% SDS, 150 mM NaCl) and cross-links were reversed overnight at 65C, followed by 1 hr RNase A and proteinase K digestion at 45 C. DNA was purified using ChIP DNA Clean & Concentrator (Zymo). For ATAC-seq samples, cells were harvested by centrifugation and 50,000 viable cells were flow sorted on ice (FACS AriaII). Cells were then washed with cold PBS and resuspended in cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630). Lysates were then incubated with Tn5 transposase at 37°C for 30 minutes. Following transposition, DNA was purified using the Qiagen MinElute PCR Purification Kit. ChIP-seq and ATAC-seq libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB).