|
| Status |
Public on Jan 10, 2017 |
| Title |
RARA-ChIP |
| Sample type |
genomic |
| |
|
| Source name |
NT2, 10μM retinoic acid for 6 days, anti-RARa
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: 10μM retinoic acid for 6 days
cell line: NT2/D1
antibody: anti-RARa
|
| Treatment protocol |
For ChIP with anti-CTCF antibodies, NT2/D1 cells were crosslinked with 1% formaldehyde at 37°C for 10 min and washed with PBS. For ChIP with anti-RARa antibodies, NT2/D1 cells were treated with dimethyl 3,3’-dithiobispropionimidate-2HCl (5 mM) (Thermo Scientific) in PBS, rinsed with an ice-cold buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl), and crosslinked with 1% formaldehyde at 37°C for 20 min. The cell pellet was resuspended in cell lysis buffer (5mM Pipes pH 8.0, 85mM KCl, 0.5%NP40 and protease inhibitors) and placed on ice for 10 min. Crude nucli fraction was pelleted by centrifugation. The pellet was resuspended in Ncli lysis buffer (50mM Tris-HCl pH8.0, 10mM EDTA 1%SDS and protease inhibitors) and incubate on ice for 10min. Lysed nucli were sonicated to generate DNA fragment of 200-1000 bp.
|
| Growth protocol |
NT2/D1 cells were cultured in Dulbecco’s modified Eagle’s minimum essential medium (DMEM; Wako) supplemented with 10% (v/v) fetal bovine serum (FBS). For retinoic acid treatment, NT2/D1 cells were treated with 10 μM all trans retinoic acid (RA; Wako)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For immunoprecipitation, chromatin samples were incubated with anti-CTCF or anti-RARa antibodies overnight at 4°C with agitation. The antibody complex were precipitated by incubation with Staph A cells at room temperature for 15 min. The supernatant was removed and the pellets were washed twice with Wash buffer A (50mM Tris-HCl pH8.0, 2mM EDTD, 0.2% Sarkosyl) and five times with Wash buffer B (100mM Tris-HCl pH 9.0, 500mM LiCl, 1% NP40, 1% deoxycholic acid). For elution, the pellets were resuspended in elution buffer (50mM NaHCO3, 1% SDS) and incubate for 15min with agitation. The supernatants were transfered to clean tubes and repeated elution.The elution samples were combined and NaCl (0.3M) and proteinase K (10μg/ml) were added and incubateed at 65°C for 5hr. The reverse crosslinked samples were purified with MinElute Reaction Cleanup Kit (QIAGEN).
|
| Label |
biotin
|
| Label protocol |
DNA was amplified by IVT (In vitro transcription) method. For the labeling, DNA was fragmented by DNaseI. DNA was end-labeled by Terminal Transferase with biotin‐N11‐ddATP
|
| |
|
| Hybridization protocol |
Samples were prepared and hybridized to GeneChip ENCODE 2.0R Array according to Affymetrix specifications.
|
| Scan protocol |
The tiling arrays were scanned using an Affymetrix GeneChip Scanner 3000 7G according to manufacturer's instruction.
|
| Data processing |
The signal intensity data was analyzed with the Affymetrix Tiling Analysis Software (v. 1.1.02) using parameters: one side upper, 250 bp bandwidth and perfect match only.
The resulting BAR files are generated by the Affymetrix Tiling Analysis Software (v. 1.1.02) using parameters: one side upper, 250 bp bandwidth and perfect match only.
|
| |
|
| Submission date |
Feb 13, 2015 |
| Last update date |
Jan 10, 2017 |
| Contact name |
Ko Ishihara |
| E-mail(s) |
kishihar@kumamoto-u.ac.jp
|
| Phone |
+81-96-373-6820
|
| Organization name |
Kumamoto University
|
| Department |
Priority Organization for Innovation and Excellence
|
| Street address |
2-2-1 Honjyo
|
| City |
Kumamoto |
| ZIP/Postal code |
860-0811 |
| Country |
Japan |
| |
|
| Platform ID |
GPL19786 |
| Series (2) |
| GSE65930 |
Removable insulator facilitates higher-order chromatin remodeling and selective gene expression in the HOXA locus via the retinoic acid signaling [ChIP-chip] |
| GSE80706 |
Removable insulator facilitates higher-order chromatin remodeling and selective gene expression in the HOXA locus via the retinoic acid signaling |
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