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| Status |
Public on Aug 07, 2019 |
| Title |
DMSO_Rep3_RNA_Seq |
| Sample type |
SRA |
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| Source name |
POP92
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| Organism |
Homo sapiens |
| Characteristics |
cell line: POP92
cell type: patient-derived colorectal cancer initiating cells
treatment: DMSO (7 days)
culture type: suspension culture
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| Treatment protocol |
For RNA-seq, cells were treated with DMSO or UNC1999 at 3uM for 7 days prior to RNA extraction
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| Growth protocol |
Cells were grown as spheroids in suspension, using cancer stem cell enriching media. Cultures were routinely passaged every 3-4 days.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were peleted and processed for RNA extraction using Rneasy (Qiagen)
For H3K27me3, cells were fixed with PBS/Formaldehyde 1%, lysed with lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1) and sonicated for 60 cycles, 30 sec ON, 30 sec OFF using the Bioruptor Plus Sonicator. For ATAC-seq, 30,000 live cells were washed with PBS and lysed for 5 minutes on ice using a lysis buffer containing 10mM Tris-HCl, pH7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630. After isolating crude nuclei, samples were treated for 30 minutes at 37ºC with Tn5 transposase, and then the DNA was purified by MinElute PCR Purification Kit. Transposed DNA fragments were amplified using specific adapters followed by purification with MinElute PCR Purification Kit.
Library construction was performed using the ThruPLEX DNA-Seq 12S Kit from Rubicon Genomics, following the manufacturer's recommendations. For ATAC-seq, Fragments from 240-360pb were selected in the PippinHT system. The quality of the library and its DNA concentration were assessed by Bioanalyzer instruments and sequenced using Illumina HiSeq 2500 sequencer, V4 chemistry.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
ChIP-Seq and ATAC-Seq were aligned to hg19 using bwa with default parameters
MACS2 was used to call peaks, with default parameters (--broad was used to call peaks in H3K27me3 and for ATAC a local background correction was applied as no input was used)
RNA-Seq was aligned using the TopHat and Cufflinks pipeline using default parameters
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-Seq data presented as bed files, RNA-Seq data as a comma separated values, cufflinks output for gene expression differences
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| Submission date |
Apr 16, 2018 |
| Last update date |
Aug 07, 2019 |
| Contact name |
Alexander James Murison |
| E-mail(s) |
alexander.murison@uhnresearch.ca
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| Organization name |
University Health Network
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| Lab |
Dick Lab
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| Street address |
101 College Street, TMDT room 11-706
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| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 1L7 |
| Country |
Canada |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE113176 |
Investigation of bivalently marked promoters in patient-derived colorectal cancer stem cells by ChIP-seq, chromatin accessibility by ATAC-seq, and differential regulation of gene expression following EZH2 inhibition using RNA-seq. |
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| Relations |
| BioSample |
SAMN08936521 |
| Supplementary data files not provided |
| Raw data not provided for this record |
| Processed data are available on Series record |
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