|Public on Feb 27, 2017
|cell type: Diffuse Intrinsic Pontine Glioma (DIPG)
passages: Low passages (6-10)
chip antibody: H3K27M (Millipore, ABE419)
|Treatment with JQ1 or DMSO was administrated only once
|Cell medium was composed as follow: DMEM 1X (Thermo Fisher), 10% serum (Corning), 1X glutamine (Life Technologies), 1X penicillin/streptomycin (Life Technologies), cells were grown in a CO2 incubator (5% CO2) at 37˚C
|RNA was extracted using QIAGEN RNeasy kit. DNA after ChIP protocol was isolated using QIAGEN PCR purification kit.
ChIP-sequencing libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250- 450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. RNA-sequencing libraries were prepared using the Illumina TruSeq Stranded Total RNA Preparation Kit with Ribo-Depletion. Input RNA quality was validated using the Agilent RNA 6000 Nano Kit. 200ng-1 µg of total RNA was used as starting material. Libraries were validated using the Agilent DNA 1000 Kit
|Illumina NextSeq 500
|Basecalls performed using bcl2fastq v18.104.22.168
Both ChIP-seq and RNA-seq reads were trimmed from 3' end until the final base had a quality score > 30, using Trimmomatic v0.33, discarding reads with < 20 bp
ChIP-seq reads were aligned to UCSC hg19 using Bowtie version 1.1.2. Only uniquely mapping reads with at most two mismatches were retained.
RNA-seq reads were aligned to UCSC hg19 using Tophat version 2.1.0. Only uniquely mapping reads with at most two mismatches were retained.
To create ChIP-seq coverage plots, the locations of the mapped ChIP-seq reads were extended to 150 bp to represent sequenced fragments, renormalized (to reads per million, rpm) and reformatted in the bigWig file format.
To create RNA-seq coverage plots, the mapped RNA-seq reads were split according to strand, renormalized (to reads per million, rpm) and reformatted in the bigWig file format.
Supplementary_files_format_and_content: bigwig files were generated using the genomicRanges R package. Score represents the normalized coverage of RNA or DNA fragments at a given genomic coordinate. RNAseq bigwigs are split by strand.
|Aug 09, 2016
|Last update date
|May 15, 2019
|Northwestern University Feinberg School of Medicine
|Department of Biochemistry and Molecular Genetics
|320 E Superior St
|Heterotypic nucleosomes and PRC2 drive DIPG oncogenesis