GSM6398633: TH-MYCN tumor, pooled input; Mus musculus; ChIP-Seq
Tissue for ChIP sequencing was snap frozen and stored in liquid nitrogen ChIP experiments and library preparation was performed by Active Motif using the HistonePath workflow. Mouse tumor tissues were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was then isolated by adding lysis buffer followed by tissue disruption with a Dounce homogenizer. Lysates were sonicated and DHA sheared to an average length of 300-500 bp. Genomic DNA (input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. DNA was quantified on a NanoDrop spectrophotometer. 30 μg chromatin was precleared with protein A agarose beads (Invitrogen) and genomic DNA regions of interest isolated using 4 μg of H3K27Ac antibody (Active Motif, cat# 39133). Complexes were washed, eluted with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 °C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Quantitative PCR reactions were carried out in triplicates on specific genomic regions using SYBR Green Supermix (Bio-Rad). Illumina sequencing libraries were prepared from ChIP and input DNA by end-polishing, dA-addition and adaptor ligation. After PCR amplification, DNA libraries were sequenced on the NextSeq 500 platform (Illumina), 75 bp reads, single-end.