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Sample GSM8395507

Query DataSets for GSM8395507

Status

Public on Jul 13, 2024

Title

Prog-Tg mouse, >25 weeks, microRNA, replicate 1

Sample type

SRA

Source name

Plasma (>25 weeks)

Organism

Mus musculus

Characteristics

tissue: Plasma (>25 weeks)
genotype: Prog-Tg
treatment: Cdh5TA+TetOp-LAG608G minigene
molecule subtype: small RNA

Treatment protocol

no treatment

Growth protocol

Endothelial cells were cultured in DMEM supplemented with 20% FCS, EC growth supplement (CellBiologics Catalog No 1166), 25 mM HEPES, 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM L-glutamine, 1 mM nonessential amino acids, 1 mM sodium pyruvate, and 139 μg/ml heparin (complete culture medium) and were used at passage 3 forpreparation of RNA extracts.

Extracted molecule

other

Extraction protocol

RNA extracts were prepared using miRNeasy Mini Kit (Qiagen 217084)
For mRNA (with parallel isolation of microRNA libraries) from endothelial cells polyA enrichment was performed for mRNAs from 100 ng total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module according to the manufacturer's instructions. Library preparation of polyA+ enriched RNA was performed with the NEBNext UltraII Directional RNA library Kit from Illumina. microRNA libraries from endothelial cells were prepared from 100 ng total RNA with the QIAseq miRNA library Kit according to the manufacturer's instructions small RNA libraries from supernatants of endothelial cells and mouse plasma were prepared using CleanTag™ Small RNA Library Preparation Kit Catalog # L-3206.

Library strategy

miRNA-Seq

Library source

other

Library selection

size fractionation

Instrument model

Illumina HiSeq 2000

Description

59991
Counts_miRNA_Plasma_Supernatant.xlsx
miRNA_Plasma_Pr_pl_vs_WT_pl.xlsx

Data processing

Sequencing reads were aligned against the mus musculus reference genome (GRCm38 release) (https://doi.org/10.1093/nar/gkx1098) with STAR (https://doi.org/10.1093/bioinformatics/bts635), version 2.5.1b using 2-pass alignment mode. Gencode v4 annotation was used in the alignment. TMM normalisation method of the edgeR (https://doi.org/10.1093/bioinformatics/btp616), R/Bioconductor package (R version 3.3.2, Bioconductor version 2.12) was applied.
The short sequencing reads were aligned against the reference genome using Gencode v4 annotation with 2-pass alignment mode of STAR v2.5.1b.
After alignment, the short reads were associated with known genes, and the number of reads aligned within each gene was counted using HTSeq v0.5.4p3.
The data was normalised to remove variation between samples caused by non-biological reasons and to make the values comparable across the sample set using the TMM normalisation method of the edgeR.
For differential expression analyses, the data were further log transformed using the voom approach, and the R package limma was used to perform the statistical testing.
Assembly: mus musculus reference genome (GRCm38 release) (https://doi.org/10.1093/nar/gkx1098)
Supplementary files format and content: Excel files starting with Counts contains the number of raw counts for each gene for each sample.
Supplementary files format and content: The rest of the Excel files (which do not start with Counts) contain differential expression results between each comparison pair using 3 replicates of each sample versus Wild Type.

Submission date

Jul 13, 2024

Last update date

Jul 13, 2024

Contact name

Roland Foisner

Organization name

Max Perutz Labs, Medical University of Vienna

Department

Center for Medical Biochemistry

Lab

Roland Foisner