Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Tumors were snap frozen and ground to a powder before fixation. Single cell suspensions were generated from cultured cells using Accutase before fixation. Samples were fixed for 5 minutes with 1% paraformaldehyde in PBS (from a frozen stock) at room temperature. Bare nuclei were suspended in shearing buffer + protease inhibitors (10 Tris HCL pH 8.0, 1 mM EDTA pH 8.0, 0.1% SDS) and sheared on a Covaris M220 ultrasonicator (Microtube, 75W, 5% duty cycle, 200 cycles per burst, 5 minutes or Millitube, 75W, 10% duty cycle, 200 cycles per burst, 15 minutes). Sonication size was verified on an Agilent Bioanalyzer using a High Sensitivity DNA Assay. For ChIP-seq, sheared chromatin from drosophila melanogaster S2 cells was added to the mammalian chromatin prior to ChIP at a set ratio for each histone mark to allow for sample normalization. 400 ng of chromatin was used per ChIP reaction and for ChIP-seq, multiple ChIPs were performed and pooled as needed to generate enough chromatin for library construction. 10-20 ng of pre-IP chromatin containing spike in was kept as an input control. ChIP reactions were performed using a modified Upstate Biotechnology protocol. Briefly, sheared chromatin was diluted 1:10 with dilution buffer + protease inhibitors (21 Tris HCL pH 8.0, 1 mM EDTA pH 8.0, 167 mM NaCl, 1.1% Triton X-100, 0.1% SDS), precleared with 30 ul Fast Flow Protein A sepharose beads (GE Healthcare) and 50 μg bovine serum albumin (BSA) by rotating in a siliconized eppendorf tube at 4 C for 1-2 hours. The precleared chromatin was transferred to a tube containing the antibody of interest (see below), 30 ul Protein A sepharose beads and 50 ug BSA and rotated overnight at 4 C. The bead bound chromatin was washed once each with low salt buffer (20 mM Tris HCl pH 8.0, 2 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS), high salt buffer (20 mM Tris HCl pH 8.0, 2 mM EDTA pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), LiCl buffer (10 mM Tris HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 1% Nonidet P-40, 1% Deoxycholate) and twice with TE (10 mM Tris HCl pH 8.0, 1 mM EDTA pH 8.0). The chromatin was eluted off the beads with 0.1 M NaHCO3 /1% SDS by rotating at room temperature for 30 minutes, then the supernatant was transferred to a tube with 200nM NaCl and cross links were reversed by incubating overnight at 65 C (input controls were processed along with the ChIP samples from this point on). 10ug Proteinase K was added and the tube was incubated for 2 hours at 37 C, then the DNA was cleaned up using a QiaQuick PCR Purification Kit (Qiagen) and eluted in 50 μl 10 mM Tris HCl pH 8.5. Antibodies used were (μl antibody per IP shown): H3K27me3 (Cell Signaling 9733, lot 8; 4 μl), H3K27ac (Cell Signaling 8173, lot 1; 4 μl) and H3K4me3 (Cell Signaling 9751, lot 8; 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).