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Sample GSM5066878

Query DataSets for GSM5066878

Status

Public on Aug 03, 2022

Title

18739T input

Sample type

SRA

Source name

Primary CRC

Organism

Homo sapiens

Characteristics

cells/tissue: tissue
chip-seq antibody: none

Extracted molecule

genomic DNA

Extraction protocol

Xenograft tumors and patient surgical specimens were digested with 1X collagenase/hyaluronidase (Stem Cell Technologies), minced, and passed through a 100μM filter. Cells were double crosslinked with 0.25M disuccinimidyl glutarate followed by 1% formaldehyde. ChIP-seq was performed as previously described3. Briefly, 2X crosslinked chromatin was sonicated on a Diagenode Bioruptor for 15 cycles 30s ON/30s OFF on the low power setting. Cells were precleared using 100μL of Magna-ChIP Protein A+G magnetic bead slurry (Millipore) before overnight incubation with 10μg ChIP antibody conjugated to100μL beads. Beads were washed once with IP Wash Buffer I, twice with High Salt Wash Buffer, once with IP Wash Buffer II, and twice with TE buffer. For buffer recipes refer to Fontanals-Cirera, Hasson et al3. Beads were eluted with 1% SDS at 65 degrees Celsius with agitation and reverse crosslinked overnight with high salt with RNase treatment (Roche). The next day, DNA underwent Proteinase K treatment at 42 degrees Celsius before elution using QIAquick PCR purification kit (QIAGEN). DNA was run on an Agilent High Sensitivity DNA chip using an Agilent Technologies 2100 Bioanalyzer for quality control. H3K27ac ChIP-seq antibodies were purchased from abcam (#ab177178). Total RNA was extracted using the QIAGEN Rneasy Mini Kit. For ChIP-seq, antibody bound DNA was purified using the QIAGEN QIAquick PCR cleanup kit.
For ChIP-seq, DNA underwent end repair, poly-adenylation, and barcoding using enzymes from New England Biolabs. ChIP libraries were amplified 12-15 PCR cycles using the 2X KAPA HiFi DNA HotStart ReadyMix before quality assessment by running on a High Sensitivity chip using a Bioanalyzer 2100 (Agilent) followed by a Qubit fluorometer (Thermo Fisher). The Mount Sinai Oncological Sciences Sequencing Core Facility prepared poly-A-capture RNA-seq libraries using the TruSeq RNA Library Prep Kit v2 (Illumina) according to manufacturer’s instructions.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

NextSeq 550

Data processing

FastQC - quality control of sequenced reads to assess duplication levels for ChIP-seq and detect adapter contamination during library prep.
cutadapt - removal of adapter contamination within raw sequenced reads
bowtie - Reads were mapped to the hg19 human genome using bowtie under the following parameters: bowtie -k 1 -m 1 --best -S -n 2 -l 60 -q --chunkmbs 200.
Samtools - Duplicate reads were removed using samtools.
MACS2 - Peak calling was performed using MACS2 with the following parameters: –f BAM –g 2.7e9 –s 75 --nomodel --extsize 200 –B –-SPMR –p 1e-10. For H3K27ac, a p-value cutoff of 10-10 was used for peak calling
deeptools - Bigwig tracks were generated using deepTools bamCoverage using the parameters: -of bigwig --effectiveGenomeSize 2736124973 --normalizeUsing RPKM
bedtools - Blacklisted regions (Duke_Hg19SignalRepeatArtifactRegions.bed, downloaded from the Broad Institute) were excluded from called peaks using bedtools
Salmon - Sequenced reads were mapped to the hg38 transcriptome using Salmon quasi-mapping under the default parameters and expression output in TPM was extracted.
Genome_build: ChIP-seq - hg19. RNA-seq hg38.
Supplementary_files_format_and_content: ChIP-seq processed data files are in the bigwig format ready for easy viewing on a genome broswer such as IGV or UCSC. RNA-seq processed data files are Salmon output excel files with raw and normalized read counts for each gene/transcript (ENSEMBL ID).

Submission date

Feb 05, 2021

Last update date

Aug 03, 2022

Contact name

Royce Zhou

E-mail(s)

[email protected]

Organization name

Icahn School of Medicine at Mount Sinai

Street address

1470 Madison Avenue