|
| Status |
Public on Jun 12, 2023 |
| Title |
OCT4-GFP positive germ cells, GR full deletion, biological rep 3 |
| Sample type |
SRA |
| |
|
| Source name |
OCT4-GFP positive germ cells sorted from E17.5 ovary
|
| Organism |
Mus musculus |
| Characteristics |
tissue: ovary
cell type: OCT4-GFP positive germ cells
developmental stage: E17.5
Sex: female
mouse strain: Tg(Pou5f1-EGFP)2Mnn Nr3c1tm1.1Jda
|
| Treatment protocol |
For both full body knockout and conditional deletion of GR, embryos were dissected from pregnant dams the morning of E17.5 into cold 0.4% BSA in PBS, and tail clips were taken from each embryo to determine GR genotype. After removing mesonephroi, fetal ovaries from individual embryos were digested for FACS sorting using 0.25% Trypsin-EDTA at 37°C for 30 min, with gentle pipetting every 10-15 minutes to facilitate dissociation. After 30 minutes, DNase I (1 mg / mL) was added at a 1:10 dilution, and samples were incubated another 10 minutes at 37°C. Samples were pipetted to ensure complete digestion, and then an equal volume of ice-cold FBS was added to inactivate trypsin. To prepare for FACS sorting, Sytox Blue viability dye was added to samples at 1:1000 dilution, and then samples were filtered through a 35 μm filter into FACS tubes. Samples were FACS sorted on a BD FACSAria II, where Tg:Oct4-GFP+ germ cells from individual embryo ovary pairs were sorted directly into QIAgen RLT+ buffer and stored at -80 until ready for RNA extractions.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was harvested using RNeasy Micro Kit (QIAgen)
Library preparations and sequencing were performed by the University of California, Davis DNA Technologies & Expression Analysis Core. Gene expression profiling was carried out using a 3’ Tag-RNA-Seq protocol. Barcoded sequencing libraries were prepared using the QuantSeq 3’ mRNA-Seq Library Prep FWD kit (Lexogen) for multiplexed sequencing according to the manufacturer recommendations. The library fragment size distribution was determined using microcapillary gel electrophoresis on a Bioanalyzer 2100 (Agilent), and libraries were quantified using a Qubit fluorometer (LifeTechnologies). Final libraries were pooled in equimolar ratios and sequenced on an Illumina HiSeq 4000.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
FFD0004
Tag-Seq_featureCounts_aggregate_female.csv
Female-DEL_vs_Female-WT.csv
|
| Data processing |
Raw fastq reads were processed to remove adapter sequences, reads containing poly-N, and low quality reads
Reads were aligned to mm10 reference genome using STAR v2.6.0
Counts of reads mapping to each gene were determined using featureCounts v1.6.3
Pairwise differential expression analysis was performed using edgeR v3.28.1 and limma v3.42.2. P-values were adjusted using Benjamini and Hochber FDR.
Assembly: mm10
Supplementary files format and content: CSV file containing raw counts for each sample
Supplementary files format and content: CSV files containing pairwise differential gene expression output from edgeR/limma
|
| |
|
| Submission date |
Jun 12, 2023 |
| Last update date |
Jun 12, 2023 |
| Contact name |
Steven Anthony Cincotta |
| E-mail(s) |
scincotta13@gmail.com
|
| Phone |
631-384-4489
|
| Organization name |
UCSF
|
| Street address |
35 Medical Center Way
|
| City |
San Francisco |
| State/province |
California |
| ZIP/Postal code |
94122 |
| Country |
USA |
| |
|
| Platform ID |
GPL21103 |
| Series (2) |
| GSE234674 |
Differential susceptibility of male and female germ cells to glucocorticoid-mediated signaling [TAGseq_KO_ALL] |
| GSE234681 |
Differential susceptibility of male and female germ cells to glucocorticoid-mediated signaling |
|
| Relations |
| BioSample |
SAMN35713463 |
| SRA |
SRX20657355 |
