|
| Status |
Public on Jul 17, 2014 |
| Title |
ChIP-Seq of Atf3 HDL+CpG BMDM (Atf3 -/-) |
| Sample type |
SRA |
| |
|
| Source name |
Atf3_HDL_CpG_KO
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
genotype/variation: Atf3 -/-
cell type: bone marrow derived macrophage
treated with: 2 mg/ml HDL for 6 h + 100nM CpG for 4 h
chip antibody: rabbit polyclonal anti-Atf3 antibody
chip antibody vendor: Santa Cruz
chip antibody cat. #: C-19X
|
| Treatment protocol |
BMDMs were pretreated with medium alone (ctrl) or 2 mg/ml HDL for 6 h or 2 mg/ml HDL for 6 h followed by stimulation with 100 nM CpG for 4 h
|
| Growth protocol |
BMDMs were obtained by culturing bone marrow cells from 6- to 8-week old WT C57BL/6 mice in DMEM supplemented with 10% (vol/vol) FCS, 10 μg/ml Ciprobay-500 and 40 ng/ml M-CSF (R&D Systems). Six days later, BMDMs were collected and plated. Age- and sex-matched WT control mice were used to derive BMDMs for comparative experiments with Atf3-deficient BMDMs. Immortalized BMDMs were cultured in DMEM supplemented with 10% FCS and 10 μg/ml Ciprobay-500 (D. De Nardo et al.; Nature Immunology 2013)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP for Atf3 was performed using the EZ-Magna ChIP G Kit (Millipore) according to the manufacturer's instructions. In brief, three biological replicates of BMDMs from WT and Atf3-deficient mice were treated with HDL, CpG, HDL then CpG or left untreated. Cells were then cross-linked with 1% paraformaldehyde, before they were lysed and chromatin was sheared by sonication for 30 min with the Covaris S220. Immunoprecipitation was performed over night at 4 °C using 2.5 μg of rabbit polyclonal anti-Atf3 antibody (C-19X, Santa Cruz). After washing, eluted samples were reverse cross-linked and treated with RNase A and proteinase K per the manufacturer's recommendations.
ChIP fragments were ligated to NEXTflex ChIP-Seq barcode adaptors using the NEXTflex ChIP-Seq Kit (both BioO Scientific). Adaptor ligated DNA fragments were size-selected (150–250 bp), PCR-amplified, further size selected (150–250 bp), pooled and sequenced on an Illumina HiSeq-1000 sequencer according to the manufacturer's instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1000 |
| |
|
| Description |
processed data file:
Atf3_HDL_CpG_KOIN_corrected_MACS_peaks_annot.txt,
Atf3_HDL_CpG_wildtype_MACS_peaks_annot.txt
|
| Data processing |
Basecalls performed using CASAVA software version 1.8
ChIP-seq reads were aligned to the NCBI Build 37 (mm9) genome assembly using Bowtie v0.12.8 with following options: -t -q -e 70 -l 28 -n 2 --best --maxbts 125 -S
After ChIP-seq quality control using HOMER v4.3, reads of biological replicates were pooled, and peaks were called using MACS v1.4.2 without Atf3-deficient BMDMs (standard method) or with Atf3-deficient dataset (KO implemented normalization = KOIN) set as background with following options: -p 1e-4 -g 1.87e9 --on-auto
Annotation of peak positions and counting of normalized Atf3 tag counts in called regions was performed with HOMER v4.3 (annotatePeaks). Peak regions with less than 2 fold change for normalized tag counts in Atf3 +/+ dataset to Atf3 -/- dataset were filtered out.
Genome_build: mm9
Supplementary_files_format_and_content: Annotated MACS peak positions in tab-delimited text format
|
| |
|
| Submission date |
Feb 25, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Joachim Schultze |
| E-mail(s) |
j.schultze@uni-bonn.de
|
| Organization name |
LIMES (Life and Medical Sciences Center Genomics and Immunoregulation)
|
| Department |
Genomics and Immunoregulation
|
| Street address |
Carl-Troll-Strasse 31
|
| City |
Bonn |
| State/province |
NRW |
| ZIP/Postal code |
53115 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15103 |
| Series (1) |
| GSE55317 |
Optimization of transcription factor binding map accuracy by utilizing their knockout-mouse models |
|
| Relations |
| BioSample |
SAMN02664584 |
| SRA |
SRX475806 |
