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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 19, 2020 |
| Title |
VEH4 |
| Sample type |
SRA |
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| Source name |
Granulosa cells
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| Organism |
Mus musculus |
| Characteristics |
treatment: vehicle
strain: C57BL/6J
cell type: Granulosa cells
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| Treatment protocol |
18-week-old female mice were injected into the peritoneum with 29 mcg of DHT suspended in a mixture of 90% sesame oil and 10% ethanol in a 100 microliter volume, and sacrificed 18 hours later. Ovaries were dissected in sterile PBS, then transferred into DMEM/F12 medium where granulosa cells were extruded by the poke-and-press method and pelleted.
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| Growth protocol |
KGN cells were maintained in DMEM/F12 supplemented with 10% fetal bovine serum and seeded into a 12-well plate to ~50% confluency, then serum starved for 24 hours before treatment with 25 nM DHT or ethanol for 12 hours.
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| Extracted molecule |
total RNA |
| Extraction protocol |
mRNA was extracted from pelleted mouse granulosa cells or cultured KGN cells using Rneasy Plus Mini Kit (Qiagen, Valencia, CA).
The total RNA concentration was determined with the NanopDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality assessed with the Agilent Bioanalyzer (Agilent, Santa Clara, CA). The TruSeq RNA Sample Preparation Kit V2 (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer’s protocols. Briefly, mRNA was purified from 100ng total RNA with oligo-dT magnetic beads and fragmented. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis. End repair and 3` adenylation was then performed on the double stranded cDNA. Illumina adaptors were ligated to both ends of the cDNA, purified by gel electrophoresis and amplified with PCR primers specific to the adaptor sequences to generate amplicons of approximately 200-500bp in size. The amplified libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina, San Diego, CA) at a concentration of 8pM per lane. Single end reads of 100nt are generated for each sample and aligned to the organism specific reference genome.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
experiment2_deSeq2_counts.txt
experiment2_deSeq2_NormCounts.txt
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| Data processing |
FastP version 0.20.0 with the following parameters: "--length_required 35 --cut_front_window_size 1 --cut_front_mean_quality 13 --cut_front --cut_tail_window_size 1 --cut_tail_mean_quality 13 --cut_tail -y -r"
STAR_2.7.0f with the following parameters: "—twopass Mode Basic --runMode alignReads --outSAMtype BAM SortedByCoordinate – outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonical –outReads UnmappedFastx"
subread-1.6.4 package (featureCounts) with the following parameters: "-s 2 -t exon -g gene_name"
Differential expression analysis was performed using DESeq2-1.22.1
Genome_build: GRCm38 + Gencode-M22 Annotation, GRChg38 + Gencode-31 Annotation
Supplementary_files_format_and_content: Raw and normalized counts
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| Submission date |
Sep 18, 2020 |
| Last update date |
Sep 20, 2020 |
| Contact name |
Cameron Baker |
| E-mail(s) |
[email protected]
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| Organization name |
University of Rochester
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| Street address |
601 Elmwood Ave
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| City |
Rochester |
| State/province |
NY |
| ZIP/Postal code |
14642 |
| Country |
USA |
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| Platform ID |
GPL21626 |
| Series (1) |
| GSE158218 |
RNA Sequencing of the Androgen-Regulated Transcriptome in Human and Mouse Granulosa Cells |
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| Relations |
| BioSample |
SAMN16209181 |
| SRA |
SRX9151825 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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