|
| Status |
Public on Sep 23, 2019 |
| Title |
NC_p53_HCT116 |
| Sample type |
SRA |
| |
|
| Source name |
HCT116
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
chip antibody: P53 (FL393, sc-6243, Lot #B1709)
treatment: Negative control siRNA transfected
|
| Treatment protocol |
HCT116 cells were treated with siRNA against iASPP, none-targeting siRNA for 3 days. And HCT116 cells were treated with Nutlin for 12 hours.
|
| Growth protocol |
HCT116 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco), L-glutamine, and appropriate antibiotics. Cell cultures were maintained in a 5% CO2 humidified incubator at 37℃.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed using the ChIP-IT High Sensitivity (HS) Kit (Active Motif 53040) according to the manufacturer’s recommended protocol with the following modifications as briefly described. Cells were cross-linked with 1% formaldehyde in DMEM culture medium for 10 min at room temperature, and quenched in 0.125 M glycine for 5 min. Cells were then washed twice with phosphate-buffered saline (PBS) followed by 3 times with lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40, 1 mM PMSF), and resuspended in ChIP Buffer (Active Motif 37516) supplemented with 1 mM PMSF and a proteinase inhibitor cocktail (Sigma-Aldrich P2714-1BTL). Chromatin was sheared by sonication (30 s alternated with 30-s cooling for 25 cycles) using a Bioruptor Pico ultrasonicator (Diagenode). Solubilised chromatin was used for immunoprecipitation by incubation overnight at 4 °C with 5 μg of the polyclonal antibody FL-393 against human p53 (Santa Cruz Biotechnology sc-6243). The immunoprecipitation reactions were transferred to Protein G Agarose Columns (Active Motif 53039) and incubated for 2 h at 4 °C. Then, each column was washed 4 times with 900 μl Wash Buffer AM1. The ChIPed samples were eluted using 100 μl Elution Buffer AM4 at 37 °C. Cross-linking was reversed by adding 5 μl Proteinase K (New England Biolabs P8107S) and incubated overnight at 65 °C. Input and ChIPed DNA were purified using the DNA purification columns.
ChIP-seq libraries were prepared using MicroPlex Library Preparation Kit v2 (Diagenode C05010014) and the Agencourt AMPure XP system (Beckman Coulter A63880) was used to select 350–550 bp libraries. Sequencing was carried out on the Illumina MiSeq and HiSeq platforms.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
All ChIP-seq reads were aligned to the hg38 reference human genome by Bowtie 2 (version 2.2.9)
Enriched p53 occupancies relative to a background input were called using MACS (version 1.4)6 with default settings.
Genomic regions with a P value < 1 × 10−10 were retained and these enriched regions were annotated using the annotatePeaks package from the HOMER suite
The bedgraph files were generated by MACS2 with the -B -trackline settings.
Genome_build: hg38
Supplementary_files_format_and_content: Genomic binding signal of p53 were illustrated as bedGraph files
|
| |
|
| Submission date |
Apr 18, 2018 |
| Last update date |
Sep 23, 2019 |
| Contact name |
Yun Tan |
| E-mail(s) |
[email protected]
|
| Organization name |
Ruijin Hospital, Shanghai Jiao Tong University School of Medicine
|
| Department |
Shanghai Institute of Hematology
|
| Street address |
Room 1109, Building 11, 197 Ruijin Er Rd
|
| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
200025 |
| Country |
China |
| |
|
| Platform ID |
GPL15520 |
| Series (1) |
| GSE113338 |
Selective and direct p53 inhibition by iASPP |
|
| Relations |
| BioSample |
SAMN08949765 |
| SRA |
SRX3962721 |
