|
| Status |
Public on Nov 05, 2014 |
| Title |
EC-5-INVASIVE |
| Sample type |
genomic |
| |
|
| Source name |
EC-5-INVASIVE
|
| Organism |
Homo sapiens |
| Characteristics |
antibody: anti-ETV5
antibody source: ETV5 (H-100)X; sc-22807X; lot#B1904; Rabbit polyclonal IgG; Santa Cruz Biotech, CA, USA
cell type: endometrial carcinoma
tumor region: invasive front
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Upon chromatin cross-linking in 1% formaldehyde, tissue from paired samples corresponding to the superficial zone and invasive front from 6 endometrial carcinomas (30mg) was disaggregated in 600μl of ChIP lysis buffer (PIC, 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS). After sonication (Branson sonifier 150; Biogen), chromatin fragments (200-1000bp) were pre-cleared with 100 μl of Protein G Agarose/Salmon Sperm DNA beads (Millipore, California, USA). Immunocomplexes of chromatin fragments and ETV5 antibodies (10 µg; Santa Cruz Biotechnologies) were recovered with G protein beads and de-cross-linked (5M NaCl at 65 °C overnight). Resulting DNA was purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany),
|
| Label |
biotin
|
| Label protocol |
Samples were labelled using the GeneChip Wtdouble-Stranded DNA Terminal Labeling Kit according to Affymetrix specifications.
|
| |
|
| Hybridization protocol |
Samples were prepared and hybridized to GeneChip Human Tiling 1.0R Array according to Affymetrix specifications.
|
| Scan protocol |
The tiling arrays were scanned using an Affymetrix GeneChip Scanner 3000 TGPLUS according to manufacturer's instruction.
|
| Data processing |
Affymetrix Human Tiling 1.0 probes were mapped to human genome assembly hg18 using corresponding BPMAP files. CEL files were imported Partek Genomic Suite 6.5 software, pre-normalized using the Robust MultichIp algorithm and converted to log2. With the purpose of detecting genes that ETV5 binds differently at the invasive front, the mean signal from each probe corresponding to the superficial zone was substracted to that of the invasion front across all probes. Statistical parameters were set as p<0.005 (paired t test).
|
| |
|
| Submission date |
May 21, 2014 |
| Last update date |
Nov 05, 2014 |
| Contact name |
Miguel Abal |
| E-mail(s) |
miguel.abal.posada@sergas.es
|
| Phone |
0034981955073
|
| Organization name |
University Clinical Hospital
|
| Department |
Medical Oncology
|
| Lab |
Translational Oncology Laboratory
|
| Street address |
Trav Choupana s/n
|
| City |
Santiago de Compostela |
| State/province |
A Coruña |
| ZIP/Postal code |
15706 |
| Country |
Spain |
| |
|
| Platform ID |
GPL5082 |
| Series (1) |
| GSE57870 |
ETV5 transcription program links BDNF and promotion of EMT at invasive front of endometrial carcinomas. |
|
