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Status |
Public on Feb 11, 2023 |
Title |
SMARCA4_786O_PBRM1WT_ChIPSeq_Rep1 |
Sample type |
SRA |
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Source name |
Cancer cell line
|
Organism |
Homo sapiens |
Characteristics |
source: 786-O (ATCC CRL1932) protein: SMARCA4 treatment: parental chip antibody: SMARCA4(AbCAM, ab110641, lot GR3208604-8)
|
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.
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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.
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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 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"
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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 |
GPL16791 |
Series (2) |
GSE152681 |
Genome-wide chromatin profiles of PBRM1 [ChIP-Seq] |
GSE152735 |
Genome-wide chromatin profiles of PBRM1 |
|
Relations |
BioSample |
SAMN15298807 |
SRA |
SRX15419259 |