|
| Status |
Public on Oct 16, 2012 |
| Title |
PrEC Cells H3K27me3 ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
PrEC
|
| Organism |
Homo sapiens |
| Characteristics |
chip antibody catalog #: sc-2003
chip antibody manufacturer: Santa Cruz
chip antibody: H3K27me3
|
| Growth protocol |
Cells were grown at 37ºC with 5% CO2 in PREGM media (Cambrex).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
~ 1 x 106 cells, in a 10cm dish, were fixed by adding formaldehyde at a final concentration of 1% and incubating for 10 minutes at 37°C. The cells were washed twice with ice cold PBS containing protease inhibitors (1mM phenylmethylsulfonyl fluoride (PMSF), 1mg/ml aprotinin and 1mg/ml pepstatin A), harvested and treated with SDS lysis buffer for 10 min on ice. The resulting lysates were sonicated to shear the DNA to fragment lengths of 200 to 500 basepairs. The complexes were immunoprecipitated with antibody specific for histone 3 lysine 27 trimethylation (Millipore #07-449). 10 ml of antibody was used. No antibody controls were also included, and no precipitation was observed by quantitative Real-Time PCR analysis. The antibody/protein complexes were collected by Protein A/G PLUS agarose beads (Santa Cruz sc-2003) and washed several times. The immune complexes were eluted with 1% SDS and 0.1 M NaHCO3 and samples were treated with proteinase K for 1 hour and DNA was purified by phenol/chloroform extraction, ethanol precipitation and resuspended in 30ml H2O. The ChIP DNA was quantitated using Qubit and 10ng was used for Illumina library preparation using Illumina ChIP-Seq DNA Sample Prep Kit #IP-102-1001.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Read files of technical replicates were pooled.
Reads were mapped with bowtie 0.12.7 Only reads mapping uniquely to the genome were kept. Upto 3 mismatches to the reference sequence were allowed. When more than one read mapped to the same genomic position, only one read was kept at that position.
Genome_build: hg18
Supplementary_files_format_and_content: BigWig. Counts across the whole genome in windows 20 bases wide, per 1 million mapped reads.
|
| |
|
| Submission date |
Jun 13, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Aaron Statham |
| E-mail(s) |
a.statham@garvan.org.au
|
| Organization name |
Garvan Institute of Medical Research
|
| Department |
Cancer Department
|
| Lab |
Epigenetics Research Laboratory
|
| Street address |
384 Victoria St
|
| City |
Darlinghurst |
| State/province |
NSW |
| ZIP/Postal code |
2010 |
| Country |
Australia |
| |
|
| Platform ID |
GPL10999 |
| Series (2) |
| GSE38683 |
H3K27me3 Marks in Normal and Cancerous Prostate Cells |
| GSE38685 |
Normal and Cancerous Prostate Cells |
|
| Relations |
| Reanalyzed by |
GSE98714 |
| SRA |
SRX154496 |
| BioSample |
SAMN01048178 |
