|
| Status |
Public on Oct 15, 2021 |
| Title |
H3K27Ac ChIP - M303_dox_neg_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
HT115
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Colorectal Cancer cells
cell line: HT115
chip antibody: H3K27ac (Diagenode C15410196 antibody)
doxycycline induction: -
expression: No doxycycline induced overexpression of V5-tagged mutant SOX9
|
| Treatment protocol |
GFP, SOX9 and mutant SOX9 was induced by doxycycline (0.5 µg/ml) for 24h. Each experiment was performed in duplicates.
|
| Growth protocol |
HT-115 cell line was obtained from Millipore Sigma and cultured in DMEM + 2mM Glutamine + 15% Fetal Bovine Serum (FBS) + 1% (P/S).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin Immunoprecipitation followed by DNA-sequencing (ChIP-seq). HT115 cells were washed with PBS and crosslinked with 1% formaldehyde for 10 minutes for H3K27ac immunoprecipitation or crosslinked with two agents starting with 2 mM DSG (Pierce) for 45 minutes at room temperature followed by 1 mL of 1% formaldehyde for 10 minutes for the V5. Cross-linked cell lines were quenched with 0.125 M glycine for 5 minutes at room temperature. Cross-linked material was resuspended in 1% SDS (50 mM Tris-HCl pH8, 10 mM EDTA) and sonicated for 5 minutes with a Covaris E220 instrument (5% duty cycle, 140 Peak Incident Power, 200 Cycles per burst, 1 mL AFA Fiber milliTUBEs). Soluble chromatin (5 µg) was immunoprecipitated with 10 μg of H3K27ac (Diagenode C15410196 antibody).
ChIP-seq libraries were constructed using Accel-NGS 2S DNA library kit from Swift Biosciences. Fragments of the desired size were enriched using AMPure XP beads (Beckman Coulter).
ChIP-Seq was performed using 36-bp paired-end reads were sequenced on a Nextseq instrument (Illumina).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Overall sequencing quality was checked with the FastQC script.
Reads were aligned to the human genome with BOWTIE using default parameters.
Peak calling was performed with MACS2 using default parameters
Genome_build: hg38
Supplementary_files_format_and_content: BigWig files were generated using deepTools
|
| |
|
| Submission date |
Oct 12, 2021 |
| Last update date |
Oct 16, 2021 |
| Contact name |
Nilay S Sethi |
| E-mail(s) |
Nilay_sethi@dfci.harvard.edu
|
| Organization name |
Dana Farber Cancer Institute
|
| Department |
Department of Medical Oncology
|
| Lab |
Gastrointestinal Cancer Research
|
| Street address |
450 Brookline Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE185736 |
An enhancer-driven stem cell-like program mediated by SOX9 blocks intestinal differentiation in colorectal cancer [h3k27ac] |
| GSE185747 |
An enhancer-driven stem cell-like program mediated by SOX9 blocks intestinal differentiation in colorectal cancer |
|
| Relations |
| BioSample |
SAMN22227005 |
| SRA |
SRX12578557 |
