|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on May 21, 2015 |
Title |
Interstitial repC |
Sample type |
SRA |
|
|
Source name |
Enriched Interstitial cells
|
Organism |
Mus musculus |
Characteristics |
strain: Sf1-eGFP tissue: gonad age: 12.5 dpc
|
Treatment protocol |
Gonads were enzymatically dissociated using 0.25% Trypsin EDTA (Gibco) or TryplE (Gibco) with 5 U/ml DNase1 (Sigma) for 20min at 37 °C and then mechanically dissociated using 18- and 23-gauge syringes. 1 mL of PBS was added to the cells which were then pelleted by centrifugation (3000 rpm at 4 °C for 10 min); after supernatant was removed the cells were resuspended in 400 μL of ice-cold PBS and stored on ice. Cells were then incubated with anti-SSEA1-PE (#FAB2155P, R&D Systems, specific for germ cells) or anti-CD31-APC (#551262, Becton Dickinson, specific for germ and endothelial cells) antibody for 20 min. The former was used in characterization of cell population studies whilst the later was used to remove germ and endothelial cells prior to RNA-Seq. Cells were fractionated using a BD FACSAria Cell Sorter. Cell populations were collected in PBS and kept on ice before further processing
|
Growth protocol |
Embryos were collected from timed matings of Sf1-eGFP strain mice (Beverdam, A. (2006)). Sf1-eGFP litters (12.5 dpc) gonads and the mesonephros were dissected in cold PBS and gonads sexed by presence of Testis cords
|
Extracted molecule |
total RNA |
Extraction protocol |
Total RNA was extracted (Micro RNeasy kit without carrier RNA, Qiagen) from CD31-treated FACS-sorted cells. Each sample represented approximately 10 sorting experiments conducted on different days with 4-10 litters of Sf1-eGFP embryos in each experiment. We prepared sample A, sample B and Sample A+B (a mix of samples A and B), with 100ng of total RNA in each sample. Libraries were prepared using TruSeq Stranded Total RNA Libraries (Illumina protocol 15031048 Rev C, Sep 2012), with multiplexing, Ribosomal depletion,with all sample run over the 4 rapid lanes
|
|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 1500 |
|
|
Data processing |
Illumina base calls Reads mapped with Tophat2 (v2.0.6), using default parameters with additional options of -G ensemble.gft --library-type=fr-firststrand --max-multihits 1 Read counts were summarized to gene features using HTSeq-count, with Ensembl mm10 gene annotation No lane effects were observed, so the four lane files per sample were merged for differential expression analysis Only genes with at least 1 count per million in three or more samples were retained for differential expression analysis Differential analyisis within R statisitcal computing enviroment (v3.0.1), normalisation with TMM and Voom, and differential analysis with Limma Genome_build: mm10 Supplementary_files_format_and_content: counts_per_Lane_per_Sample.txt: Raw counts of sequencing reads for the gene features, per lane per sample. Supplementary_files_format_and_content: Interstitial_vs_Sertoli.txt, Leydig_vs_Sertoli.txt, Leydig_vs_Sertoli.txt: Differential analysis results extracted from the limma analysis.
|
|
|
Submission date |
Feb 02, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Katrina M Bell |
E-mail(s) |
katrina.bell@mcri.edu.au
|
Phone |
61399366709
|
Organization name |
MCRI
|
Street address |
RCH, Flemington Rd, Parkville
|
City |
Melbourne |
State/province |
Victoria |
ZIP/Postal code |
3052 |
Country |
Australia |
|
|
Platform ID |
GPL18480 |
Series (1) |
GSE65498 |
Purification and transcriptomic analysis of mouse fetal Leydig cells reveals candidate genes for disorders of sex development |
|
Relations |
BioSample |
SAMN03323794 |
SRA |
SRX862826 |
Supplementary data files not provided |
SRA Run Selector |
Processed data are available on Series record |
Raw data are available in SRA |
|
|
|
|
|