 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 30, 2024 |
| Title |
sw480_smad4_bir_TAGCTT.june2015 |
| Sample type |
SRA |
| |
|
| Source name |
SW480
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SW480
cell type: colorectal cancer (CRC)
|
| Treatment protocol |
Cells stably expressing both avi-tagged SMAD4 and BirA were selected with medium containing 2 μg/ml puromycin and 0.4 mg/ml Hygromycin B, respectively.
|
| Growth protocol |
SW480 cells were cultured in RPMI (Gibco), containing 10% FBS (Gibco) and 1% penicillin and streptomycin (Gibco).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Each 100 μl cell pellet was incubated with 1 ml of cross-linking solution containing an equal amount of DSG (Thermo), DSS (Thermo) and EGS (Thermo 21565), creating a final concentration of 2 mM NHS ester (reactive groups). Cells were rocked for 20 min at room temperature, after which fresh formaldehyde was added to make a concentration of 1.22% formaldehyde. The cells were then rocked for an additional 20 min. The cross-linking reaction was stopped with 125 mM glycine. The cells were then washed with PBS, and nuclei were extracted using a Dounce homogenizer and Farnham lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, 0.5% NP-40, and protease inhibitor cocktails). IP material was subsequently handled as described above for conventional ChIP of sonicated genomic DNA (a final concentration of 0.2% SDS in the sonicates). The diluted sonicates were then incubated with pre-blocked (0.5% BSA/PBS) Streptavidin beads (Invitrogen) overnight at 4°C. The beads were serially washed in low salt (0.1% SDS, 0.1% Sodium Deoxycholate, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, and 20 mM HEPES, pH 8.0), high-salt (0.1% SDS, 0.1% Sodium Deoxycholate, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, and 20 mM HEPES, pH 8.0), LiCl buffer (250 mM LiCl, 0.5% Sodium Deoxycholate, 0.5% NP-40, 1mM EDTA, and 20 mM HEPES, pH 8.0), and a final wash buffer (1 mM EDTA and 20 mM HEPES, pH 8.0). The beads were then incubated at 65 °C for 6 h in reverse cross linking buffer (0.1 mM NaHCO3 and 1% SDS). The DNA was purified by QIAquick PCR Purification Kit (QIAGEN) and quantified with PicoGreen (Life Technologies).
5 ng each of ChIP or input DNA was used to prepare ChIP-seq libraries with a Rubicon Genomics ThruPLEX DNA-seq Kit (Illumina).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
FastQC (v.0.11.3) was used to check the quality of raw sequencing reads (fastq), and bowtie2 (v.2.2.6) was used to align the sequences to mouse (mm9) or human (hg19) genomes and generate bam files. Deeptools bamCoverage (v.2.4.2, duplicate reads ignored, RPKM normalized and extended reads) was used to generate bigwig files from bam files. Model-based Analysis of ChIP-Seq (MACS 1.4.1) was used for peak calling and to generate bed files from aligned reads. The shiftsize parameter used in MACS was based on the fragment size of Pippin Prep. SMAD4 ChIP-seq of SW480 cells are at a P value of 10−5.
Assembly: hg19
Supplementary files format and content: txt file, MACS summit centered peaks
|
| |
|
| Submission date |
May 10, 2023 |
| Last update date |
May 30, 2024 |
| Contact name |
Kevin Tong |
| E-mail(s) |
kevin.tong@hmh-cdi.org
|
| Organization name |
Hackensack Meridian Health
|
| Department |
CDI
|
| Street address |
111 Ideation Way
|
| City |
Nutley |
| State/province |
NJ |
| ZIP/Postal code |
07110 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE232136 |
In vitro organoid-based assays reveal SMAD4 tumor-suppressive mechanisms for serrated colorectal cancer invasion [ChIP-seq] |
| GSE232138 |
In vitro organoid-based assays reveal SMAD4 tumor-suppressive mechanisms for serrated colorectal cancer invasion |
|
| Relations |
| BioSample |
SAMN35021412 |
| SRA |
SRX20276732 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
