|
| Status |
Public on Oct 30, 2013 |
| Title |
LN4D6 CREB3L1 HA ChIP |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
CREB3L1 (HA) ChIP DNA from LN4D6 CREB3L1 cells
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: LN4D6
cell type: rat mammary tumor cells
transfected with: CREB3L1
chip antibody: HA-X (F-7) antibody
chip antibody vendor: Santa Cruz
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin Immunoprecipitation (ChIP) assay was performed using an EpiQuick™ Chromatin Immunoprecipitation Kit (Epigentek™) and a HA-X (F-7) antibody (Santa Cruz). All procedures were carried out according to the manufacturer’s specifications. The ChIP DNA was then amplified using a GenomePLex Whole Genome Amplification Kit -4 (WGA-4) (Sigma), according to the manufacturer’s specifications. The amplified DNA was then purified using a QIAquick® PCR purification kit (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy5 or Cy3 nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| |
|
| Channel 2 |
| Source name |
Input DNA from LN4D6 CREB3L1
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: LN4D6
cell type: rat mammary tumor cells
transfected with: CREB3L1
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin Immunoprecipitation (ChIP) assay was performed using an EpiQuick™ Chromatin Immunoprecipitation Kit (Epigentek™) and a HA-X (F-7) antibody (Santa Cruz). All procedures were carried out according to the manufacturer’s specifications. The ChIP DNA was then amplified using a GenomePLex Whole Genome Amplification Kit -4 (WGA-4) (Sigma), according to the manufacturer’s specifications. The amplified DNA was then purified using a QIAquick® PCR purification kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy5 or Cy3 nonamers per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| |
|
| |
| Hybridization protocol |
The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)
|
| Scan protocol |
Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
|
| Description |
Chip-chip LN4D6 CREB3L1 cells HA (CREB3L1)
|
| Data processing |
Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction software at Nimblegen.
The ratio_peaks.gff is the ratio of signal for each transcript on the chip between the ChIP sample and the input, which is used to determine if a positive signal is unique to the ChIP sample. The 'ratio_peaks_mapToFeatures_All_Peaks.txt' file contains data with the identity of the feature on it e.g. name and accession number.
|
| |
|
| Submission date |
Oct 28, 2013 |
| Last update date |
Oct 30, 2013 |
| Contact name |
Deborah Anderson |
| E-mail(s) |
deborah.anderson@saskcancer.ca
|
| Phone |
13069667038
|
| Organization name |
University of Saskatchewan
|
| Street address |
107 Wiggins Road
|
| City |
Saskatoon |
| State/province |
Saskatchewan |
| ZIP/Postal code |
S7N 5E5 |
| Country |
Canada |
| |
|
| Platform ID |
GPL17848 |
| Series (2) |
| GSE51802 |
Association of CREB3L1 with promoters in LN4D6 CREB3L1 cells |
| GSE51857 |
CREB3L1 is a metastasis suppressor that represses expression of genes regulating metastasis, invasion and angiogenesis |
|
