|
| Status |
Public on Oct 09, 2019 |
| Title |
VRTN_ChIPSeq |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
cell type: Human Embryonic Kidney
passages: 15-18
chip antibody: Anti-FLAG M2 Affinity Gel (Sigma)
|
| Treatment protocol |
The full-length coding sequence of pig VRTN was subcloned into pNTAP-2×flag expression vectors. HEK293T cells were transfected with pNTAP-2×flag-VRTN.
|
| Growth protocol |
HEK293T cells were cultured in high-glucose DMEM (Gibco) supplemented with 10% fetal bovine serum (Hyclone) and an antibiotic solution
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP samples were prepared from VRTN-overexpressing HEK293T cells using the SimpleChIP Enzymatic Chromatin IP kit (CST) according to the manufacturer’s instructions. The cells were harvested and chemically cross-linked with 1% formaldehyde for 10 minutes at 16-20°C. The cell nuclei were released via sonication using an Ultrasonic Processor (SONXI). The collected nuclei were digested with micrococcal nuclease (CST) for 20 minutes at 37 °C. Sheared chromatin was incubated with Anti-FLAG M2 Affinity Gel (Sigma) for 4 hours. Immunoprecipitated DNA was treated with RNase A (CST) and purified with the SimpleChIP kit (CST).
A sequencing library was constructed from enriched DNA fragments using the TruSeq ChIP Library Prep kit (Illumina). Final DNA libraries were validated and sequenced at 50 bp per sequence read using a HiSeq 2000 sequencer (Illumina) at a depth of approximately 20 million sequences.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Data processing |
ChIP-seq reads were mapped to the human genome (GRCh38.p5) using BEDtools.
Only the alignments with fewer than two mismatches were considered for enriched-region (peak) identification (peak calling) using the Model-based Analysis of ChIP-seq (MACS) algorithm.
MEME 4.6.1 software was used to identify the VRTN binding motif.
PeakAnalyzer software was used for peak annotation and the analysis of the nearest downstream gene.
Genome_build: the human genome (GRCh38.p5)
Supplementary_files_format_and_content: wig files were generated with MACS algorithm.
|
| |
|
| Submission date |
Oct 09, 2016 |
| Last update date |
Oct 09, 2019 |
| Contact name |
Yanyu Dean |
| E-mail(s) |
yanyuduan@gmail.com
|
| Phone |
86-791-83813080
|
| Organization name |
Jiangxi Agricultural University
|
| Lab |
State Key Laboratory of Pig Genetic Improvement and Production Technology
|
| Street address |
Lushanzhong888, Changbei district
|
| City |
Nanchang |
| ZIP/Postal code |
330044 |
| Country |
China |
| |
|
| Platform ID |
GPL9052 |
| Series (2) |
| GSE87774 |
VRTN Regulates the Number of Thoracic Vertebrae During Vertebrate Development [ChIP-seq] |
| GSE87776 |
VRTN Regulates the Number of Thoracic Vertebrae During Vertebrate Development |
|
| Relations |
| BioSample |
SAMN05891564 |
| SRA |
SRX2234299 |
