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Sample GSM1598869

Query DataSets for GSM1598869

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