GSM4728431: lucap49_AS1; Homo sapiens; ChIP-Seq
Nuclei isolated as previously described were crosslinked with 1% Formaldehyde for 10 minutes for H3K27Ac ChIP-seq and crosslinked in 2 steps with 2 mM of DSG (Pierce) for 45 minutes at room temperature followed by 1 ml of 1% Formaldehyde for 10 minutes whereas for ASCL1 and NEUROD1. Crosslinked nuclei were then quenched with 0.125 M glycine for 5 minutes at room temperature and washed with PBS. After fixation, pellets were resuspended in 500 ul of 1% SDS (50 mM Tris-HCl pH 8, 10 mM EDTA) and sonicated in 1 ml AFA fiber millitubes for 5 minutes (H3K27ac) or 10 (ASCL1 and NEUROD1) minutes using a Covaris E220 instrument (setting: 140 peak incident power, 5% duty factor and 200 cycles per burst). Chromatin was immunoprecipitated with 10 ug of H3K27Ac antibody (Diagenode cat# C15410196), 10 ug of ASCL1 antibody (abcam ab74065) or 10 ug of NEUROD1 antibody (Cell Signalling mAb #4373). 5 ug of chromatin was used for H3K27Ac ChIPs, and 40 ug of chromatin was used for ASCL1 or NEUROD1 ChIPs. ChIP-seq libraries were made using Rubicon kit (reference) and purified. 75-bp single-end reads were sequenced on a Nextseq instrument (Illumina).