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Status |
Public on Jan 12, 2019 |
Title |
Day14.5-C |
Sample type |
SRA |
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|
Source name |
Extracellular vesicles - GD14.5 pregnant mouse sera
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Organism |
Mus musculus |
Characteristics |
strain/background: C57BL/6 sample origin: ExoQuick isolated extracellular vesicles from mouse sera pregnancy status: GD14.5
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Treatment protocol |
Gestational day (GD) 0.5. Pregnant female mice were sacrificed on GD 14.5. Age-matched, non-pregnant females (NP) were sacrificed in conjunction with each pregnant female. At time of sacrifice (by carbon dioxide asphyxiation followed by bilateral pneumothorax), blood was collected via cardiac puncture. Serum was prepared from the whole blood using venous blood collection serum separator tubes (BD Vacutainer #367986) per manufacturer’s instructions. Serum was stored at -80°C.
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Growth protocol |
Murine TSCs derived from C57BL/6 mice were maintained on CellStart-CTS coated plastic plates in RPMI-1640 with L-glutamine (Hyclone SH30027) supplemented with 1% penicillin/streptomycin (Hyclone SV30010), 1% sodium pyruvate (Gibco 11360), 85.8 µM β-mercaptoethanol (Bio-Rad 161-0710), 1 μg/ml heparin (Sigma H3149), 25 ng/ml fibroblast growth factor 4 (FGF4) (Sigma F8424) to maintain stemness (STEM; samples 1-6). TSCs cultured on plastic in the absence of FGF4 or heparin at normal oxygen tension (O2, 21%) result in fused, multinuclear syncytial-like cultures (SynT).
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Extracted molecule |
total RNA |
Extraction protocol |
RNA was purified from ExoE-EV using SeraMir RNA kits (System Biosciences). RNA-Seq libraries were constructed using modified Illumina adapter methods using the XMIR exosome RNA-Seq Sample Preparation Kit (System Biosciences) and indexed with separate bar codes for multiplex sequencing on an Illumina MiSeq v3 instrument using a 2 x 75 bp paired end run setting.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina MiSeq |
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Description |
Illumina MiSeq v3 processed data file: Abundances_Day14.5.txt processed data file: NPvsDay14.5_all_with_type.1384366252915.tsv
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Data processing |
Data quality check of the input sequence was first performed using FastQC. Bowtie2 was then used to map the spike-in DNA before the trimming (ea-utils and cutadapt) and filtering (PRINSEQ) steps. FastQC was re-run to analyze the trimmed reads, allowing a before and after comparison. Sequence reads were merged using SeqPrep then mapped to the reference genome using Bowtie, followed by generation of a mapping rate summary. Using the open-source software BEDTools and SAMtools, read alignment and read coverage tracks were generated and deployed to the customized UCSC genome browser. Quantitation and alignment to miRBase. Abundance determination and Abundance levels for ncRNAs (miRNAs, tRNAs, rRNAs, lincRNAs, piRNAs, and snoRNAs), antisense transcripts, coding genes and repeat elements (LTR, LINE, SINE, and tandem repeats) were determined. A summary of reads overlapping each of these annotations in the reference genome was created using SAMtools. Genome_build: mm10 (GRCm38) Supplementary_files_format_and_content: Abundances_Day14.5.txt: Abundances for Day14.5 samples (tab-delimited text). Supplementary_files_format_and_content: Abundances_Diff.txt: Abundances for Diff samples (tab-delimited text). Supplementary_files_format_and_content: Abundances_NP.txt: Abundances for NP samples (tab-delimited text). Supplementary_files_format_and_content: Abundances_Stem.txt: Abundances for Stem samples (tab-delimited text). Supplementary_files_format_and_content: Differences in expression of ncRNA were calculated and differential expression analysis was performed in R using DESeq; adjusted P-values were calculated using Benjamini-Hochberg procedure. Differential expression results are provided for each comparison: 1) NP vs. Day 14.5 (NPvsDay14.5_all_with_type.1384366252915.tsv) and 2) Stem vs Diff (StemvsDiff_all_with_type.1384366250358.tsv).
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Submission date |
Jan 11, 2019 |
Last update date |
Jan 13, 2019 |
Contact name |
Virginia D. Winn |
E-mail(s) |
vwinn@stanford.edu
|
Organization name |
Stanford University
|
Department |
OB-GYN/Reproductive, Perinatal and Stem Cell Biology Research
|
Street address |
300 Pasteur Drive, Boswell A364
|
City |
Stanford |
State/province |
CA |
ZIP/Postal code |
94305 |
Country |
USA |
|
|
Platform ID |
GPL16417 |
Series (1) |
GSE124995 |
Next-generation sequencing of murine trophoblast-derived and pregnancy-associated exosome-enriched extracellular vesicle microRNAs |
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Relations |
BioSample |
SAMN10734412 |
SRA |
SRX5243541 |