|
| Status |
Public on May 09, 2015 |
| Title |
Bre12p8_ChIP_input |
| Sample type |
genomic |
| |
|
| Source name |
vHMEC, none
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Breast
passage: 8
|
| Treatment protocol |
~2x10^6 cells (per immunoprecipitation) fixed for 10 minutes at 37°C in culture media containing 2.7% (w/v) formaldehyde. Media was discarded and cells were washed in ice cold PBS, then scraped in cold PBS and pelleted and stored at -80C until required.
|
| Growth protocol |
HMEC lines were generated from tissue taken from healthy women with informed donor consent as part of breast reduction mammoplasty (institutional ethics approval provided by the Sydney Adventist Hospital Ethics Committee, Project ID 06/03). At the time of surgery, all donors were aged between 23 and 43 years of age. Breast tissue was diced and then digested with collagenase to generate organoids. To establish HMEC lines, organoids were allowed to expand in MBD170 serum free basal medium (Life Technologies, Carlsbad CA, USA) until large colonies (1-2 cm) were observed (5-10 days). Organoid colonies were partially trypsinised to remove stromal cells and the remaining epithelial cells were transferred into a new tissue culture flask to generate passage 1 HMEC. vHMEC lines were then generated by serial passage of HMEC until proliferation slowed and the cells entered the selection phase. Selection phase HMEC were maintained with twice-weekly media changes until large, actively growing colonies (1-2 cm) were observed. Growing cells were trypsinised and transferred to a new culture vessel, generating the vHMEC lines.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell peeltes were resuspended in SDS cell lysis solution (Millipore) and IP performed using the millipore Chromatin Immunoprecipitation (ChIP) Assay Kit using 10ug of antibody (Anti-H3K27me3 or anti-H3K9ac)
|
| Label |
Biotin
|
| Label protocol |
DNA was amplified using the whole genome amplification (Genomeplex WGA2 Kit, Sigma Aldrich). WGA material was cleaned-up with the GeneChip® Sample Cleanup Module (Affymetrix, Santa Clara CA, USA) and 7.5 μg was then fragmented and labelled for array hybridisation with the GeneChip® WT Double-Stranded Target Assay (Affymetrix, Santa Clara CA, USA) according to the manufacturer’s protocol.
|
| |
|
| Hybridization protocol |
Hybridisation was performedt according to the conditions set ou by Affymetrix
|
| Scan protocol |
Scan was performedt according to the conditions set ou by Affymetrix
|
| Description |
Formalin Fixed 10min 37C + Sonicated to ~500bp mean fragment size
Fixed Chromatin
|
| Data processing |
Low level analysis and Model-based Analysis of Tiling arrays (MAT) normalisation of ChIP-chip data was performed using the R package aroma.affymetrix. Array signal across all promoters on the array was determined using the R package Repitools. ChIP data was then summarised to a t-statistic for a region of ±2 kb of all TSS with the Repitools function “blocksStats”
|
| |
|
| Submission date |
Jun 27, 2014 |
| Last update date |
May 09, 2015 |
| Contact name |
Warwick James Locke |
| Organization name |
The Garvan Institute of Medical Research
|
| Department |
Cancer Deparment
|
| Lab |
Epigenetics Research Program
|
| Street address |
384 Victoria St
|
| City |
Darlinghurst |
| State/province |
NSW |
| ZIP/Postal code |
2010 |
| Country |
Australia |
| |
|
| Platform ID |
GPL5082 |
| Series (2) |
| GSE58880 |
Coordinated epigenetic remodelling occurs during early breast carcinogenesis [Human promoter ChIP-chip] |
| GSE58882 |
Coordinated epigenetic remodelling occurs during early breast carcinogenesis |
|
