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Sample GSM5464791 Query DataSets for GSM5464791
Status Public on Feb 04, 2022
Title Infected (deltaE) - 2dpi - Rep3 [E2D RNA-seq]
Sample type SRA
Source name Infected (deltaE)_2dpi_lung
Organism Mus musculus
Characteristics strain: BALB/c OlaHsd
age: 16-week-old
infected with: rSARS-CoV-ΔE
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 E2D_CGTACG
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
State/province Madrid
ZIP/Postal code 28049
Country Spain
Platform ID GPL13112
Series (1)
GSE180563 Contribution of host miRNA-223-3p to SARS-CoV-induced lung inflammatory pathology
BioSample SAMN20342123
SRA SRX11515189

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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