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Sample GSM2785495

Query DataSets for GSM2785495

Status

Public on Jan 25, 2018

Title

RNA-seq of mouse liver + LNA-negative_6 days_female1_G153_M8

Sample type

SRA

Source name

live_LNA-negative_6 days_female

Organism

Mus musculus

Characteristics

strain: Crl:CD1(ICR)
age: 10 weeks
Sex: Female
treatment: Scrambled LNA (negative control) for 6 days
tissue: liver

Biomaterial provider

Charles River; strain code #022

Treatment protocol

To inhibit miR-1948-5p and miR-802-5p function in mouse liver in vivo, LNA constructs complementary to these miRNAs and a negative control were purchased from Exiqon and injected into male and female 9 wk ICR mice (mmu-miR-1948-5p:GGCAGAATACTCATA; mmu-miR-802-5p: GAATCTTTGTTACTG; negative control: ACGTCTATACGCCCA). Mice were given two subcutaneous injections of the LNA in 1X PBS solution at 25 mg/kg on Day 0 and again on Day 1. Mice were killed and euthanized on Day 3 or Day 6, to give a total of three days or six days of LNA exposure in vivo. Injections were carried out in the morning between 10 am - 12 pm. LNA-miR-1948 was injected into male mice; LNA-miR-802 was injected into female mice; LNA-negative control was injected into both male and female mice for both 3 day and 6 day exposures, and served as controls. At the end of the 3 day or 6 day treatments, mice were killed by cervical dislocation, and livers were harvested for RNA extraction and RNA-seq library construction from poly(A) enriched mRNAs. For each sex and at each time point, N=5 mice received LNA-anti-miRNA, and N=3 mice received scrambled LNA (negative controls).

Extracted molecule

total RNA

Extraction protocol

RNA was extracted from flash fozen mouse liver using Trizol per the manufacturer's instructions.
RNA was depleted of rRNA using the NEBNext Poly(A) mRNA Magnetic isolation module (NEB, Ipswich, MA, Cat# E7490S). Libraries were prepared with the NEBNext Ultra Directional RNA Library Prep kit for Illumina (NEB, Cat# E7420) and multiplexed using NEBNext Multiplex Oligos for Illumina (NEB, Cat# E7335, E7500), according to the manufacturer's instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Sequence reads were mapped to the mouse genome (mm9) using TopHat2 (Trapnell et al, Nat. Biotechnol, 2010)
FeatureCounts (Liao, 2014, Bioinformatics) was used to obtain read counts for RefSeq genes. Mature (spliced) mRNAs were quantified by counting sequence reads that map to a concatenated sequence comprised of the union of the exonic regions for all isoforms of a given gene. Primary RNA transcripts (hnRNA) were quantified by counting reads that map to the intersection of all intronic sequences across all isoforms of a given gene; it thus excludes sequences found in an exonic region of at least one isoform of the gene.
Differential expression analysis for RefSeq genes was conducted using edgeR (Robinson, 2010, Bioinformatics),
Genome_build: mm9
Supplementary_files_format_and_content: Microsoft Excel file containing normalized read counts and 12 pair-wise differential expression results.

Submission date

Sep 14, 2017

Last update date

May 15, 2019

Contact name

David J. Waxman

E-mail(s)

djw@bu.edu

Organization name

Boston University

Department

Department of Biology and Bioinformatics Program