|
| Status |
Public on Nov 21, 2016 |
| Title |
EBF1-Wt-ChIP |
| Sample type |
SRA |
| |
|
| Source name |
A-MuLV transformed pro-B cells (Ebf1fl/fl;RERTCre), 2μM 4-hydroxy-tamoxifen (4-OHT) treated
|
| Organism |
Mus musculus |
| Characteristics |
cell type: A-MuLV transformed pro-B cells (Ebf1fl/fl;RERTCre)
strain: C57BL/6
|
| Treatment protocol |
A-MuLV transformed Ebf1fl/fl RERTCre pro-B cells were transfected by pmys-EBF1wt-IRES-GFP or pmys-EBF1-H240A-IRES-GFP and treated with 2μM 4-hydroxy-tamoxifen (4-OHT) 24h after transduction. Five days after treatment, cells were sorted as GFP+. More than two week later, the replacement was validated by IB and the cells were used for downstream analyses. As control, mock-transduced cells were harvested four days after tamoxifen treatment before cell death was observed.
|
| Growth protocol |
A-MuLV transformed cells were cultured without feeder cells in RPMI medium supplemented with 10% FCS, 1% PSG and 50μM β-mercaptoethanol.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with formaldehyde, lysed and DNA was fragmented into pieces of average 300bp by sonication. Chromatin was precipitated with a specific antibody and protein A-coupled sepharose beads. After elution from the beads, chromatin was decrosslinked and purified via QIAquick PCR Purification Kit according to the manufacturer's manual.
Library preparation for ChIP analysis was performed with the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (Catalog #E7370) according to the manufacturer’s protocol. No size selection was used and amplification was performed using 10 PCR cycles.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Genome-wide EBF1-wt occupancy was performed by replacement approach
|
| Data processing |
Basecalling and fastq files generation was performed by using Illumina bcl2fastq 1.8.4 (RTA 1.18.64)
The reads were aligned to the mm9 genome assembly using bowtie2 with default configurations
Duplicated reads were filtered and blacklisted regions were removed using samtools and Picard.
Peaks were identified using MACS2 (v2.1.0.20150731) with q-value cutoff 0.01 and further filtered for p-value 10-7 and fold change 4
Genome_build: mm9
Supplementary_files_format_and_content: The processed bedgraph files were generated by using Homer(v4.6) with default parameters. The reads are normalized to 10 million reads.
|
| |
|
| Submission date |
Oct 05, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Senthilkumar Ramamoorthy |
| E-mail(s) |
senthilkumar@ie-freiburg.mpg.de
|
| Phone |
00497615108731
|
| Organization name |
Max Planck Institute of Immunobiology and Epigenetics
|
| Department |
Cellular & Molecular Immunology
|
| Lab |
Laboratory Rudolf Grosschedl
|
| Street address |
Stübeweg 51
|
| City |
Freiburg |
| ZIP/Postal code |
D-79108 |
| Country |
Germany |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE87636 |
Genome-wide occupancy of EBF1-wt & EBF1-H240A |
| GSE87637 |
Interaction of CNOT3 with EBF1 regulates gene-specific transcription and mRNA stability in B lymphopoiesis |
|
| Relations |
| BioSample |
SAMN05863805 |
| SRA |
SRX2212166 |
