|
| Status |
Public on May 28, 2015 |
| Title |
UW203, cKIT+ RNA Seq Ovaries |
| Sample type |
SRA |
| |
|
| Source name |
Fetal Ovary
|
| Organism |
Homo sapiens |
| Characteristics |
sample: embryo
age: 67 days
cell type: cKIT+ FACS sort
|
| Treatment protocol |
Human fetal gonads, H1 and UCLA1 hESCs were dissociated with 0.25% Trypsin-EDTA, supplemented with 2% chicken serum (Gibco BRL), for 5 min at 37oC. Dissociated cells were incubated in 1% BSA in PBS containing primary antibodies on ice for 20 min. Primary antibodies used were cKIT-APC conjugated (BD Biosciences) and TRA-1-81 (eBioscience).
|
| Growth protocol |
Human embryos were obtained following elected termination and pathological evaluation at the Laboratory of Developmental Biology at the University of Washington, Seattle. All consented material was anonymous and carried no personal identifiers. The BDRL estimates developmental age by prenatal intakes, foot length, Streeter’s stage and crown-rump length. Any conceptus with a documented birth defect or chromosomal abnormality was excluded from our study. Human ESCs line H1 and UCLA1 were maintained by culture under self –renewal conditions on mouse embryonic fibroblast (MEF) layer in DMEM:F12 (Gibco BRL), 20% KnockOut Serum (Gibco BRL), 1% nonessential amino acids (NEAA, Gibco BRL), 1 mM L-glutamine (Gibco BRL), 0.1 mM β-mercaptoethanol (Gibco BRL), and 10ng/ml of basic fibroblast growth factor (FGF) from R&D.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were sorted directly in 75ul RLT buffer (Qiagen) and total RNA was extracted using the RNeasy Micro Kit (Qiagen) according to manufacturer’s instructions.
RNA Seq libraries were genreated using the Ovation RNA-Seq V2 (Nugen) followed by Encore Rapid Library System (Nugen) according to manufacturers instructions.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Reads with low quality, and reads containing sequencing adapters were first filtered out.
Raw reads were mapped to hg19 with the gapped aligner Tophat2 allowing up to two mismatches.
Transcript expression levels were quantified using RPKM units using customized scripts written in Perl after normalization based on the geometric means as described in DESeq.
For the transposon analysis, repeat annotation file of h19 was downloaded from the UCSC genome browser (http://genome.ucsc.edu/). The RPKM values of repeats were calculated according to coding transcripts.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text file include RPKM values for each sample.
|
| |
|
| Submission date |
Nov 18, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Sofia Gkountela |
| E-mail(s) |
sgkountela@ucla.edu
|
| Organization name |
UCLA
|
| Department |
MCDB
|
| Lab |
Amander Clark
|
| Street address |
621 Charles E Young Dr S
|
| City |
Los Angeles |
| State/province |
California |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE63392 |
RNA-Seq of cKIT+ sorted cells from 53-137 day old fetal testes and ovaries and RNA-Seq of TRA-1-81+ H1 and UCLA1 hESCs. |
| GSE63394 |
cKIT+ sorted cells from 57-137 day old fetal testes and ovaries |
|
| Relations |
| BioSample |
SAMN03199088 |
| SRA |
SRX761429 |
