|
| Status |
Public on Aug 31, 2015 |
| Title |
ATF7_pMp_Whole_F |
| Sample type |
genomic |
| |
|
| Source name |
Peritoneal macrophages from wild-type mice
|
| Organism |
Mus musculus |
| Characteristics |
treatment: ChIP using ATF7 Ab
strain: wild-type in C57BL/6 background
cell-type: peritoneal macrophage
|
| Treatment protocol |
Unterated
|
| Growth protocol |
Peritoneal resident macrophages were collected by peritoneal washing, without thioglycollate injection, and were incubated in Macrophage-SFM (Life Tech.) containing 100 U/ml penicillin and 100 µg/ml streptomycin. Before and after overnight incubation, dish surfaces were washed by pipetting with PBS to remove non-adherent cells such as lymphocytes.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Peritoneal macrophages were crosslinked with 1% formaldehyde for 8 min at RT and after washing with ice-cold PBS prepared for ChIP. Chromatin immunoprecipitation was performed using ATF7 antibody. The lysate was incubated with the antibodies over night at 4 degree, and then protein A dynabeads (Life Tech.) were added and incubated for 3 hours at 4 degree. The beads were washed several times and eluted with elution buffer (50 mM Tris, 10 mM EDTA, 1 % SDS). After reverse cross-linking by overnight incubation at 65 degree, the eluate from the beads was treated with Proteinase K. Contaminating RNA was removed by RNAse treatment. The sample was further purified by phenol-chloroform extraction and using the PCR purification 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 |
Each sample was hybridized to the array in 200 ul containing 1XHybridization buffer (Affymetrix), 50pM Control oligonucleotide (oligo B2, Affymetrix), Herring Sperm DNA (0.1mg/ml), Acetylated BSA (0.5mg/ml), and 7% DMSO. Samples were denatured at 100C for 10 minutes, and then put on ice before being hybridized for 16 hours at 45C in an hybridization oven (GeneChip Hybridization Oven 640, Affymetrix). Washing and scanning protocol provided by Affymetrix was performed automatically on a fluidics station (GeneChip fluidics station 450, Affymetrix).
|
| Scan protocol |
Arrays were scanned using the Genechip Scanner3000 7G following the library array description.
|
| Description |
tiling_ATF7_MAT_signalNorm.bed
ATF7 distribution in whole genome of peritoneal resident macrophage. Array F of A-G
|
| Data processing |
We calculated Model-Based Analysis of Tiling-Arrays (MAT) score software implementation in R (rMAT package). Signals were mapped against mouse genome assembly mm8 (NCBI Build 36), and ChIP was normalized against input for both Mouse Tiling 2.0R Arrays and Mouse Promoter 1.0R Arrays. Normalization and MAT score computations were performed using default parameters (Bandwidth = 600; MaxGap = 300; and MinProbe = 8) and the ‘pair-binned’ method for normalization. Enriched regions (peaks) were then detected using a p-value threshold set to 10-10 for both arrays. All detected peaks have a false-discovery rate, as computed by rMAT, lower than 1%.
The resulting BED files are generated by rMAT using default parameters (Bandwidth = 600; MaxGap = 300; and MinProbe = 8). Enriched regions are then detected using a p-value threshold set to 10-10 for both arrays.
|
| |
|
| Submission date |
Jul 20, 2015 |
| Last update date |
Aug 31, 2015 |
| Contact name |
Keisuke Yoshida |
| E-mail(s) |
keiyoshida@rtc.riken.jp
|
| Organization name |
RIKEN
|
| Lab |
Molecular Genetics Laboratory
|
| Street address |
3-1-1 Koyadai,
|
| City |
Tsukuba |
| State/province |
Ibaraki |
| ZIP/Postal code |
305-0074 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6449 |
| Series (2) |
| GSE71112 |
ATF7 mediates lipopolysaccharide-induced epigenetic changes in macrophages involved in innate immunological memory (ChIP) |
| GSE71113 |
ATF7 mediates lipopolysaccharide-induced epigenetic changes in macrophages involved in innate immunological memory |
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