title: GSM3192302: X7a-12_Ctl_H3K27ac_ChIP-seq; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP and library construction for SJDIPGX7 xenografts using H3K27me3 (Millipore 07-449), H3K27ac (Active Motif 39133) and H3K4me3 (Active Motif 39159) was performed by Active Motif.  All other ChIP was performed at St. Jude using the following method.  Xenograft tumors were snap frozen and ground to a powder before fixation.  Single cell suspensions were generated from cultured cells then fixed. For histone ChIP, samples were fixed for 5 minutes with 1% paraformaldehyde in PBS (from a frozen stock) at room temperature.  For BMI1 and RNF2 ChIP, samples were fixed with 2mM disuccinimidyl glutarate in PBS for 45 minutes at room temperature followed by 1% paraformaldehyde in PBS for 15 minutes at room temperature following the method established in Gargiulo et al. (van Lohuizen lab, 2013 Cancer Cell 23:660-676). Bare nuclei were sheared on a Covaris M220.  For histone mark ChIPs, the reads arising from mouse cells entrapped within the xenograft to normalize the post ChIP samples to the human/mouse read ratio obtained for the pre-ChIP material (input library).  For some experiments, chromatin from drosophila S2 cells was also spiked-in and used to validate the use of mouse reads for normalization.  ChIP reactions were performed using a modified Upstate Biotechnology protocol. Antibodies used were (μl antibody per IP shown): H3K27me3 (Cell Signaling 9733, lot 8; 4 μl), H3K27ac (Cell Signaling 8173, lot 1; 4 μl), H3K4me3 (Cell Signaling 9751, lot 8; 5 μl), BMI1 (Bethyl A301-694A, lot 4; 5 μl) and RNF2 (Bethyl A302-869A, lot 1; 5 μl). Libraries were prepared from 5-10 ng of DNA using the NEBNext ChIP-Seq Library Prep Reagent Set for Illumina with NEBNext Q5 Hot Start HiFi PCR Master Mix according to the manufacturer's instructions (New England Biolabs) with the following modifications: a second 1:1 Ampure cleanup was added after adaptor ligation. The Ampure size selection step prior to PCR was eliminated. Completed libraries were analyzed for insert size distribution on a 2100 BioAnalyzer High Sensitivity kit (Agilent) or Caliper LabChip GX DNA High Sensitivity Reagent Kit (Perkin Elmer).  Libraries were quantified using the Quant-iT PicoGreen dsDNA assay (Life Technologies), Kapa Library Quantification kit (Kapa Biosystems) or low pass sequencing on a MiSeq Nano v2 run (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM3192302