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Status |
Public on Aug 22, 2017 |
Title |
A-498 H3K27ac ChIP-seq |
Sample type |
SRA |
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Source name |
ccRCC cell line
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Organism |
Homo sapiens |
Characteristics |
histone: H3K27ac chip antibody: H3K27ac (ab4729, Abcam) source: A498 (ATCC HTB-44)
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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.
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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.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 2500 |
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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"
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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 |
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Relations |
BioSample |
SAMN05710548 |
SRA |
SRX2055056 |