|
| Status |
Public on Aug 22, 2017 |
| Title |
40911432 H3K27ac ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
ccRCC cell line
|
| Organism |
Homo sapiens |
| Characteristics |
histone: H3K27ac
chip antibody: H3K27ac (ab4729, Abcam)
source: in house derived
|
| Growth protocol |
All ccRCC cells (786-O, A498 and tumor-derived cell lines) were grown in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum (Gibco). HK-2 and PCS400 were grown in Renal Epithelial Cell Basal Medium (ATCC). Cells were maintained in a 5% CO2-humidified incubator at 37°C. In order to derive cell lines from primary tumors, ccRCC primary tumor tissues were disassociated by collagenase. Dissociated cells were maintained in RPMI with 10% FBS. At 80-90% confluency, the cells were passaged at 1:3 ratio. The cultured cells were considered to be successfully immortalized after 60 passages.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
One million cells were fixed in 1% formaldehyde/PBS buffer for 10 min at room temperature. Fixation was stopped by addition of glycine to a final concentration of 125 mM. Cells were washed 3 times with TBSE buffer. Pelleted cells were lysed in 100 µl 1% SDS lysis buffer and sonicated to 300-500bp using a Bioruptor (Diagenode). The total volume of immunoprecipitation was 1 ml and the amount of antibody used was 2 µg. The input DNA was precleared with protein G Dynabeads (Life Technologies) for 1 hr at 4°C and then incubated with antibodies conjugated protein G beads overnight at 4°C. The beads were washed 3 times with cold wash buffer.
Whole-genome-amplification was performed on ChIP DNA and input using the WGA4 kit (Sigma-Aldrich). The amplified DNA was used with NEBNext ChIP-Seq library prep reagent set.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Sequencing reads were trimmed (10bp from front and back) and 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 CCAT (p-value < 0.05).
Genome_build: hg19
Supplementary_files_format_and_content: bed format for ucsc genome browser, including 5 columns: "chromosome" "start of region" "end of region" "peak name" "fold change"
|
| |
|
| Submission date |
Aug 26, 2016 |
| Last update date |
May 15, 2019 |
| 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 |
GPL16791 |
| Series (2) |
| GSE86087 |
cis-regulatory landscape in ccRCC [cell lines] |
| GSE86095 |
VHL deficiency drives enhancer activation of oncogenes in clear cell renal cell carcinoma |
|
| Relations |
| BioSample |
SAMN05710565 |
| SRA |
SRX2055037 |
