|
| Status |
Public on Dec 01, 2014 |
| Title |
VCaP Input DNA [ChIP-Seq control] |
| Sample type |
SRA |
| |
|
| Source name |
VCaP cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Prostate cancer cell line
passages: 3 to 4
chip antibody: none (input)
|
| Treatment protocol |
For estrogen treatment experiment, VCaP and NCI-H660 cells were hormone starved for 48 hours prior to treatment with 10nM of 17 b estradiol (E2) for 14 hours
|
| Growth protocol |
Prostate cell lines were maintained in appropriate medium. VCaP and VCaP ERa expressing cells were maintained in DMEM (Invitrogen) and supplemented with 10% fetal bovine serum (FBS) with 1% penicillin streptomycin. NCI-H660 cells were grown in RPMI 1640 supplemented with 0.005 mg/ml insulin, 0.01 mg/ml transferrin, 30 nM sodium selenite, 10 nM hydrocortisone, 10 nM 17 beta estradiol, 5% FBS and an extra 2 mM of L-glutamine (for a final concentration of 4 mM).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was fixed in 1% formaldehyde, sheared to 200-700bp using an ultrasonic sonicator (Brandson), immunoprecipitated using the indicated antisera, purified and sequenced using Illumina Genome Analyzers as recommended by the manufacturer.
Libraries were prepared according to Illumina's instructions accompanying the ChIP-Seq Sample Kit (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 15 cycles and library fragments from 200-300 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries for VCaP + E2 and NCI H660 + E2 were sequenced on the Genome Analyzer II and for VCaP cells, VCaP ERa + E2 and Sample Input in a HiSeq 2000.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Sequencing reads were aligned to the reference genome (hg18) using Illumina GERALD (both NCI H660 cell lines and VCaP + E2) or CASAVA 1.8 (VCaP and VCaP ERα) using default parameters
Peak detection for all ChIP-seq experiments was performed with ChIPseeqer (Giannopoulou and Elemento, 2011), using the same parameters for all datasets (i.e., p-value threshold for peaks=10-5, minimum distance between peaks=100bp). For details, please go to: http://icb.med.cornell.edu/wiki/index.php/Elementolab/ChIPseeqer_use
Genome_build: hg18
Supplementary_files_format_and_content: BED files of the ERa binding sites locations for the different cell lines
|
| |
|
| Submission date |
Feb 01, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Dimple Chakravarty |
| E-mail(s) |
dic2012@med.cornell.edu
|
| Phone |
(646)962-6189
|
| Organization name |
Weill Cornell Medical College
|
| Department |
Pathology
|
| Lab |
BB 1450
|
| Street address |
Belfer Research Building 413 E 69 St., New York
|
| City |
NEW YORK |
| State/province |
NY |
| ZIP/Postal code |
10021 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE43985 |
The Estrogen receptor alpha regulated NEAT1 long non-coding RNA promotes prostate cancer progression [ChIP-Seq] |
| GSE43988 |
The Estrogen receptor alpha regulated NEAT1 long non-coding RNA promotes prostate cancer progression |
|
| Relations |
| SRA |
SRX222276 |
| BioSample |
SAMN01909123 |
