|
| Status |
Public on Sep 30, 2014 |
| Title |
input DNA |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic stem cell culture
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: embryonic stem cells
chip antibody: N/A
|
| Growth protocol |
Mouse ESC cultures, N6 (WT) and N8 (REST -/-) (Jorgensen et al., 2009), were cultured in DMEM supplemented with 1000 units/ml Leukemia Inhibitory Factor (LIF, ESGRO from Millipore), 15% fetal bovine serum (FBS), 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1X non-essential amino acids (NEAA), 1X nucleoside mixture (adenosine, guanosine, uridine, cytidine, and thymidine), and 1X penicillin/streptomycin. ESCs were cultured on feeder layers of irradiated mouse embryonic fibroblasts and passaged three times on plates coated with 0.1% gelatin to eliminate MEFs before harvesting cells for chromatin preparations.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Nuclei isolations were lysed and sonicated to shear chromatinbefore immunopurification of crosslined DNA/protein complexes.
ChIP-isolated DNA was pooled (three technical replicates done in parallel from each of two independent biological replicates) and fragments were processed to blunt ends followed by A-tailing to facilitate ligation of Illumina oligo adapters. PCR amplification was run for 12-14 cycles with primers complementary to adapter sequence to amplify the pool of ChIP DNA with addition of the adapter sequence. PCR products in the range of 200-300 bp were isolated by agarose gel electrophoresis followed by gel extraction.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
Illumina software pipeline was used for base-calling.
BWA was used to align sequence reads to mm9 mouse genome assembly and reads with less than 4 individual base mismatches that aligned to a single unique location were filtered for further analysis.
Peak analysis of REST-binding was independently performed using Peak Ranger and MACS analysis software to identify regions of enrichment relative to Input control and overlapping peaks were retained.
Wiggle file were produced using default settings of the PeakRanger wigmodule
Genome_build: mm9
|
| |
|
| Submission date |
Jun 26, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Gail Mandel |
| Organization name |
Oregon Health & Science Universtiy
|
| Department |
Vollum Institute
|
| Lab |
Gail Mandel
|
| Street address |
3181 SW Sam Jackson Park Rd.
|
| City |
Portland |
| State/province |
OR |
| ZIP/Postal code |
97239 |
| Country |
USA |
| |
|
| Platform ID |
GPL9250 |
| Series (1) |
| GSE48320 |
Polycomb Repressor Complex 2 and REST-associated histone deacetylases are independent pathways toward a mature neuronal phenotype. |
|
| Relations |
| BioSample |
SAMN02214037 |
| SRA |
SRX315202 |
