|
| Status |
Public on Dec 31, 2015 |
| Title |
I/11 cells 96 h after infection with anti-Plcg1 shRNA #14; 24h on differentiation medium |
| Sample type |
SRA |
| |
|
| Source name |
murine erythroblast cell line
|
| Organism |
Mus musculus |
| Characteristics |
cell line: I/11
|
| Treatment protocol |
For methylome analysis, I/11 cells were infected with lentiviral particles (by spinfection; 2000 rpm for 1.5 hours at 33°C) containing either control or Plcγ1-specific shRNAs. After 24 hours of infection cells were cultured in serum-free medium (StemPro®34) supplemented with 1 U/ml of human recombinant Erythropoietin (Epo), 100 ng/ml SCF, 1 µM dexamethasone and 1% penicillin/streptomycin for 24 hours, followed by selection with puromycin (1 µg/ml) for 48 hours. For differentiation induction, I/11 were seeded 72 hours after infection in StemPro34® medium containing 10 U/ml Epo, 0.5 mg/ml human holo-transferrin and 1% penicillin/streptomycin. Cells were harvested at the indicated time points (0h on differentiation; 24h on differentiation).
|
| Growth protocol |
The expanding erythroid progenitors (I/11) were maintained in serum-free medium (StemPro®34 plus nutrient supplement) supplemented with 1 U/ml of human recombinant Erythropoietin (Epo), 100 ng/ml SCF, 1 µM dexamethasone and 1% penicillin/streptomycin. For differentiation induction I/11 cells were seeded in StemPro34® medium containing 10 U/ml Epo, 0.5 mg/ml human holo-transferrin and 1% penicillin/streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated using Qiagen DNA mini kit. 3 μg of genomic DNA were sonicated in a Covaris S sonicator. DNA fragments which were about 150 bp in size were then subjected to enrichment with 60 μg MBD2-Fc protein in the presence of a salt gradient. The DNA fractions highly enriched for methylated DNA (eluted with 1 M NaCl) were subjected to next-generation sequencing.
NEBNext ChIP-seq Library Prep Reagent Set for Illumina
|
| |
|
| Library strategy |
MBD-Seq |
| Library source |
genomic |
| Library selection |
MBD2 protein methyl-CpG binding domain |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
small hairpin RNA against Plcg1
I/11 cells is a murine erythroblast cell line established from p53-deficient mouse fetal liver cells (day E12.5)
|
| Data processing |
Sequence reads were aligned to the mouse genome assembly mm10 using BWA with default parameters and only uniquely aligned reads were kept for further analysis.
PCR-duplicates were identified and removed using picard and samtools with default parameters.
Peak calling was done with the Homer software package with a fixed window size of 150 bp and otherwise default settings using the scrambled shRNA as a reference.
Genome_build: mm10
Supplementary_files_format_and_content: Peak file (BED format)
|
| |
|
| Submission date |
Aug 05, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Daniel B. Lipka |
| E-mail(s) |
[email protected]
|
| Organization name |
German Cancer Research Center
|
| Department |
Epigenomics and Cancer Risk Factors
|
| Street address |
INF 280
|
| City |
Heidelberg |
| ZIP/Postal code |
69120 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE60087 |
Epo-Induced Erythroid Maturation Is Dependent on Plcγ1 Signaling |
|
| Relations |
| BioSample |
SAMN02951832 |
| SRA |
SRX669396 |
