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Sample GSM5464801

Query DataSets for GSM5464801

Status

Public on Feb 04, 2022

Title

Infected (Wild Type) - 2dpi - Rep3 [W2E RNA-seq]

Sample type

SRA

Source name

Infected (Wild Type)_2dpi_lung

Organism

Mus musculus

Characteristics

strain: BALB/c OlaHsd
age: 16-week-old
infected with: rSARS-CoV wild type
time point: 2dpi
tissue: lung

Treatment protocol

16-week-old mice were inoculated with rSARS-CoV wt or rSARS-CoV-ΔE in 50 μl of DMEM containing 2% fetal bovine serum (FBS, Biowhittaker). Mice were sacrificed at days 2 and 4 post infection and lung and serum samples were collected.

Growth protocol

Specific-pathogen-free 8 week-old BALB/c OlaHsd female mice were purchased from Harlan. Mice were maintained for 8 additional weeks in the animal care facility at the National Center of Biotechnology (CNB-CSIC, Madrid).

Extracted molecule

total RNA

Extraction protocol

To isolate long and small RNAs separately, one-half of the right lung was homogenized in 2 mL of Lysis/Binding Solution (mirVana miRNA Isolation Kit, Ambion) using a MACS homogenizer (Miltenyi Biotec), according to manufacturer’s protocols. Small and long RNAs were extracted separately from total RNA samples using mirVana miRNA Isolation protocol.
.6 µg of the long RNA fraction were treated to eliminate ribosomal RNA (RiboZero rRNA Removal Kit, Epicentre) and libraries were constructed (NEBNext Ultra directional Library prep A, Illumina) and subjected to 100SE sequencing on a Hiseq 2000 sequencer (Illumina, Parque Científico de Madrid, PCM), resulting in 20 million reads per sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

W2E_GAGTGG

Data processing

Raw 100nt strand-specific single-end reads were quality checked with FASTQC. As many samples presented undetermined nucleotides (“N”) and decreased quality after 76th position, all reads from all samples were truncated to the first 75 nucleotides.
Truncated (75nt) reads were aligned against mouse genome sequence (GRCm38) with TopHat2 (Kim et al. 2013) allowing intron sizes up to 100.000 nt and using default settings for single-end alignments.
Htseq-count function of HTSeq package (Anders et al., 2014) was used for assigning reads to each mouse gene and lncRNA. Genomic feature coordinates were obtained from mouse annotation version GRCm38.76 (ENSEMBL).
To evaluate differential expression of features between SARS-CoV-∆E (2dpi) and WildType (2dpi) samples, DESeq2 (Love et al., 2014) was used. Default parameters were used except detection of outlier counts by Cook’s distance that was not applied (cooksCutoff=FALSE). Three biological replicates per sample were included in the analysis and p.values obtained were adjusted by FDR (Benjamini and Hochberg, 1995).
Genome_build: GRCm38
Supplementary_files_format_and_content: tab-delimited text file include raw read count values for all samples

Submission date

Jul 21, 2021

Last update date

Feb 04, 2022

Contact name

Juan Carlos Oliveros

Organization name

CNB, CSIC

Street address

Darwin 3

City

Cantoblanco