|
| Status |
Public on Jul 01, 2011 |
| Title |
SMAD4 unstimulated cell ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
A2780 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A2780 cells
treatment: TGF-beta
chip antibody: SMAD4 (20% Cell Signaling Technology, 80% Santa Cruz Biotechnology)
antibody manufacturer 1: Cell Signaling Technology
antibody manufacturer 2: Santa Cruz Biotechnology
antibody catalog number 1: 9515
antibody catalog number 2: sc-7359
antibody lot number 1: 2
antibody lot number 2: H0408
antibody number 1 fraction: 20%
antibody number 2 fraction: 80%
|
| Treatment protocol |
80% confluant cells were stimulated with 10ng/ml recombinant TGFβ1 for 1 hour prior to formaldehyde crosslinking.
|
| Growth protocol |
cells grown in RPMI 1640 supplemented with 10% FBS in 5% CO2 incubator at 37C. Cells were inspected daily and split at ~70% confluance.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and SMAD4-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions accompanying the ChIP-seq DNA Sample prep Kit (Part# IP-102-1001). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 12 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were isolated from a 2% E-gel from invitrogen. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Chromatin IP against SMAD4 in SMAD4 unstimulated cells
|
| Data processing |
hg18, peak files produced using BELT
|
| |
|
| Submission date |
Feb 25, 2011 |
| Last update date |
Jun 11, 2013 |
| Contact name |
Brian Alexander Kennedy |
| E-mail(s) |
kennedy.642@osu.edu
|
| Organization name |
The Ohio State University
|
| Street address |
278 Chittenden AVE
|
| City |
Columbus |
| State/province |
Ohio |
| ZIP/Postal code |
43201 |
| Country |
USA |
| |
|
| Platform ID |
GPL10999 |
| Series (1) |
| GSE27526 |
ChIP-seq defined genome-wide map of TGFbeta/SMAD4 targets: implications with clinical outcome of ovarian cancer patients |
|
| Relations |
| BioSample |
SAMN02197053 |
