|
| Status |
Public on May 16, 2019 |
| Title |
129_Cast_ESC_Sox2_SCR_Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: F1 hybrid 129/Castaneus
dev_stage: Embryonic stem cell
Sex: Female
cell_line: 129/CastEiJ F1 hybrid
cell_type: Mouse embryonic stem cell
genotype: Sox2-8CcuO/+; Sox2-117TtetO/+
|
| Growth protocol |
Embryonic stem cells were maintained in 2i + Lif media and grown at 37ºC with 5% CO2. Cells were passaged every 2 days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with 1% formaldehyde in PBS for 10 minutes at room temperature. Nuclei were isolated in lysis buffer (10mM Tris-HCl pH8.0, 10mM NaCl, 0.2% Igepal CA630, 1X protease inhibitor). Chromatin was digested with 100 U of DpnII overnight at 37ºC. Next, samples were ligated with 2000 U of T4 DNA ligase for 4 hours at room temperature. After ligation, samples were incubated in 10% SDS with 1 mg/mL Proteinase K, follwed by reverse crosslinking through addition of 5M NaCl and incubating at 65ºC overnight. DNA was then linearized with 50U NlaIII overnight at 37ºC.
200ng of 4C template was used for PCR amplification for 30 cycles and 3-4 reactions were pooled together. Primers were designed to include Illumina adaptor sequences as well as barcodes derived from Illumina's TruSeq adaptors. PCR products were then purified using dual SPRI bead selection to select 120-1000bp fragments. Library concentrations were quantified using the KAPA qPCR system.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Library strategy: 4C-seq
Reads were trimmed of the reading primer sequences using cutadapt (DOI: 10.14806/ej.17.1.200) with the following call: cutadapt -g
Reads were mapped to the mm9 genome using bowtie2 (PMID: 22388286) with default settings
BigWig files were created using deepTools (PMID: 27079975) with the following call: deepTools bamCoverage -bs 1
Genome_build: mm9
Supplementary_files_format_and_content: bigWig files for 4C-seq read density aligned to mouse genome
|
| |
|
| Submission date |
Mar 06, 2019 |
| Last update date |
May 18, 2019 |
| Contact name |
Jeffrey Michael Alexander |
| E-mail(s) |
jeffrey.alexander@ucsf.edu
|
| Organization name |
Gladstone Institutes
|
| Lab |
Bruneau Lab
|
| Street address |
1650 Owens St
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE127901 |
4C on Sox2 Locus with tetO/cuO Modifications |
|
| Relations |
| BioSample |
SAMN10985705 |
| SRA |
SRX5406240 |
