GSM4404962: mIUE_G34R-WT_4_H3K27me3; Mus musculus; ChIP-Seq
For ChIP-seq, cells were fixed with 1% formaldehyde (Sigma). Fixed cell preparations were washed, pelleted and stored at -80°C. Sonication of lysed nuclei (lysed in a buffer containing 1% SDS) was performed on a BioRuptor UCD-300 for 60 cycles, 10s on 20s off, centrifuged every 15 cycles, chilled by 4°C water cooler. Samples were checked for sonication efficiency using the criteria of 150-500bp by gel electrophoresis. After the sonication, the chromatin was diluted to reduce SDS level to 0.1% and before ChIP reaction ~5% of sonicated drosophila S2 cell chromatin was spiked-in the samples for quantification of total levels of histone mark after the sequencing (see below). ChIP reaction for histone modifications was performed on a Diagenode SX-8G IP-Star Compact using Diagenode automated Ideal ChIP-seq Kit. 25ul Protein A beads were washed and then incubated with 6ug of H3K27ac antibody (Diagenode, C15410196), and 2 million cells of sonicated cell lysate combined with protease inhibitors for 10 hr, followed by 20 min wash cycle with provided wash buffers. Reverse cross linking took place on a heat block at 65°C for 4 hr. ChIP samples were then treated with 2ul RNase Cocktail (Life Technologies) at 65°C for 30 min followed by 2ul Proteinase K (Thermo Fisher Scientific) at 65°C for 30 min. Samples were then purified with QIAGEN MiniElute PCR purification kit as per manufacturers' protocol. In parallel, input samples (chromatin from about 50,000 cells) were reverse crosslinked and DNA was isolated following the same protocol. For RNA-seq, total RNA was extracted from cell pellets and mouse tumors using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen) according to instructions from the manufacturer. Library preparation was performed with ribosomal RNA (rRNA) depletion according to instructions from the manufacturer (Epicentre) to achieve greater coverage of mRNA and other long non-coding transcripts. Paired-end sequencing was performed on the Illumina HiSeq 2500/4000 platform. For Hi-C, libraries were generated from 5 million cultured G34 glioblastoma cell lines KNS-42, HSJD-GBM002 and PS10-801 as described in (Rao et al., 2014) with minor modifications. Briefly, in situ Hi-C consists of 7 steps: (1) crosslinking cells with formaldehyde, (2) digesting the DNA using a 4-cutter restriction enzyme (e.g., DpnII) within intact permeabilized nuclei, (3) filling in, biotinylating the resulting 5′-overhangs and ligating the blunt ends, (4) shearing the DNA, (5) pulling down the biotinylated ligation junctions with streptavidin beads, (6) library amplification and (7) analyzing these fragments using paired end sequencing. As quality control (QC) steps, we checked for efficient sonication with an agarose DNA gel and for appropriate size selection using the Agilent Bioanalyzer on final amplified libraries. For the final QC, we performed superficial sequencing on the Illumina Hiseq 2500 (~30M reads/sample) to assess quality of the libraries using percent of reads passing filter, percent of chimeric reads, and percent of forward-reverse pairs.  125bp paired-end sequencing was performed on the Illumina HiSeq 4000. For ChIP-seq, library preparation was carried out using Kapa HTP Illumina library preparation reagents. Briefly, 25ul of ChIP sample was incubated with 45ul end repair mix at 20°C for 30 min followed by Ampure XP bead purification. A tailing: bead bound sample was incubated with 50ul buffer enzyme mix for 30°C 30 min, followed by PEG/NaCl purification. Adaptor ligation: bead bound sample was incubated with 45ul buffer enzyme mix and 5ul of different TruSeq DNA adapters (Illumina) for each sample, for 20°C 15 min, followed by PEG/NaCl purification (twice). Library enrichment: 12 cycles of PCR amplification. Size selection was performed after PCR using a 0.6x/0.8x ratio of Ampure XP beads (double size selection) set to collect 250-450bp fragments. Single-end or paired-end 50bp sequencing was performed on the Illumina HiSeq 4000 or NovaSeq 6000 platform. For RNA-seq, RNA-seq library preparation was performed with ribosomal RNA (rRNA) depletion according to instructions from the manufacturer (Epicentre) to achieve greater coverage of mRNA and other long non-coding transcripts, paired-end sequencing (100 bp) was performed on the Illumina HiSeq 4000 platform.
GEO Accession
GSM4404962