|
| Status |
Public on Feb 11, 2023 |
| Title |
H3K27ac_786O_PBRM1WT_ChIPSeq |
| Sample type |
SRA |
| |
|
| Source name |
Cancer cell line
|
| Organism |
Homo sapiens |
| Characteristics |
source: 786-O (ATCC CRL1932)
protein: H3K27ac
treatment: parental
chip antibody: H3K27ac (AbCAM, ab4729, lot 741806)
|
| Treatment protocol |
PBRM1 was knocked out using CRISPR-Cas9 mediated gene editing in 786-O, A498 and HK-2. Wildtype PBRM1 was cloned from normal kidney tissue. cDNA was either cloned into pbabe vector or pLenti-GFP.
|
| Growth protocol |
Cells were grown in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum (Gibco). Cells were maintained in a 5% CO2-humidified incubator at 37°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each protein of interest, approximately 2x107 cells were cross-linked with 1% formaldehyde for 10 min at room temperature, and stopped by adding glycine to a final concentration of 0.2M. Chromatin was extracted and sonicated to ~500bp (Vibra cell, SONICS). The total volume of immunoprecipitation was 2 ml and the amount of antibody used was 20 µg. The input DNA was precleared with protein G Dynabeads (LifeTechnologies) for 2 hours at 4°C and then incubated with antibodies overnight at 4°C. Protein G beads were added the following day and mixture was nutated for 3 hours at 4°C. The beads were washed 6 times with wash buffer at room temperature.
At least 10 ng of the amplified DNA was used with NEBNext ChIP-Seq library prep reagent set (NEB). Each library was sequenced to an average depth of 30-50 million reads on HiSeq2500 or HiSeq4000.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Sequencing reads were mapped against human genome reference hg19 using the Burrows-Wheeler Aligner (BWA) (version 0.6.2) 'mem' algorithm. Only reads with mapQ >10 and with duplicate removed by rmdup were used in the subsequent analysis
Significant peaks were called using MACS2 24 (q-value < 0.01).
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-seq peaks are in bed format, including 5 columns: "chromosome" "start of region" "end of region" "peak name" "peak enrichment"
|
| |
|
| Submission date |
Jun 17, 2020 |
| Last update date |
Feb 17, 2023 |
| Contact name |
Xiaosai Yao |
| E-mail(s) |
xiaosai.yao@gmail.com, yao.xiaosai@gene.com
|
| Phone |
+65 68088271
|
| Organization name |
Genome Institute of Singapore
|
| Street address |
60 Biopolis Street, Genome, #02-01
|
| City |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE152681 |
Genome-wide chromatin profiles of PBRM1 [ChIP-Seq] |
| GSE152735 |
Genome-wide chromatin profiles of PBRM1 |
|
| Relations |
| BioSample |
SAMN15298805 |
| SRA |
SRX15419261 |
