|
| Status |
Public on Dec 31, 2011 |
| Title |
SP1 ChIP-seq - HCT116 |
| Sample type |
SRA |
| |
|
| Source name |
SP1 ChIP-seq - HCT116
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
antibody: SP1
antibody vendor: Active Motif
antibody catalog number: #39058
antibody lot number: 19108005
|
| Growth protocol |
Human colon adenocarcinoma cell line HCT116 (ATCC) was maintained in McCoy 5A Media Modified (Gibco) supplemented with 10% fetal bovine serum (Gibco). To select for PB transposition, G418 was added to the media to a final concentration 700 mg/ml. Cells were cultured at 37 oC in the presence of 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Calling Card Illumina libraries were prepared by harvesting G418 resistant cells and extracting their genomic DNA. Each DNA sample was divided into three aliquots and digested with Taq1 or Csp61 or Msp1. Digested DNA was ligated overnight at 15°C in dilute solution (<10ng/ul) to encourage self-circularization. After ethanol precipitation, self-ligated DNA was resuspended in ddH2O and used as template in an inverse PCR. Primers that anneal to PB Donor sequence (OM8721 and OM8722) were used to amplify the genomic regions flanking PB integrations and the bar code within the transposon, as well as adding adapter sequences that allow the PCR products to be sequenced on the Illumina GA IIx analyzer. The PCR products were purified using the QIAquick PCR Purification Kit (Qiagen) and diluted to 10nM. For each sample, the same amount of PCR product from digestion with each restriction endonuclease was pooled and submitted for sequencing on the Illumina GAII.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
SP1 ChIP-seq
|
| Data processing |
SP1_ChIPseq_peaks.bed; genome build: hg18
For Calling Card insertions: Paired reads were filtered by requiring the first bases to contain the end of the PB TR (to ensure specific amplification) on the first read, and a proper barcode and digestion site on the second read. The non-genomic sequences were trimmed and the remaining sequence from each paired-end read was aligned to hg18 using bowtie with the options ‘--best --tryhard --minins 0 --maxins 1200’. Reads that could be uniquely aligned counted as an independent insertion for every position for every unique barcode at that position. For ChIP-seq data: Model-based analysis of ChIP-seq (MACS) was used to generate the peaks file. For RNA-seq data: reads were aligned to hg18 using Tophat and transcripts were quantified using Cufflinks.
bowtie version 0.12.7
MACS version 1.4.0alpha
cufflinks version 1.1.0
|
| |
|
| Submission date |
Dec 30, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
David Mayhew |
| E-mail(s) |
david.n.mayhew@gsk.com
|
| Organization name |
GlaxoSmithKline
|
| Department |
Target Sciences, Computation Biology, R&D
|
| Street address |
1250 South Collegeville Road
|
| City |
Collegeville |
| State/province |
PA |
| ZIP/Postal code |
19426 |
| Country |
USA |
| |
|
| Platform ID |
GPL10999 |
| Series (1) |
| GSE34791 |
"Calling Cards" for DNA-binding proteins in mammalian cells |
|
| Relations |
| SRA |
SRX113706 |
| BioSample |
SAMN00769283 |
