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Sample GSM5820454 Query DataSets for GSM5820454
Status Public on Oct 19, 2022
Title Ferret lung_air control_rep1 [air_209_F2]
Sample type SRA
Source name Ferret lung_air control
Organism Mustela putorius furo
Characteristics Sex: female
treatment: exposed to room air
batch: 2
tissue: Lung tissue
Extracted molecule total RNA
Extraction protocol RNA isolated from snap frozen lung tissue using Qiagen miRNeasy Mini Kit  Cat No./ID: 217004 and later we did ribosome reduction for samples.  We used the NEBNext rRNA Depletion kit (Human/Mouse/Rat), cat E7400.
RNA libraries were prepared for sequencing using standard Illumina protocols
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Data processing RNA-seq data for 12 samples (six biological replicates each for air and smoke experimental conditions) were sequenced using an Illumina NextSeq 500 System. Pre-alignment quality assessments of the raw fastq sequences were carried out using FastQC (v0.11.7) (Andrews, 2010). The number of paired ends reads for the twelve samples range from 35M to 57M. The raw fastq sequences were aligned to the Mustela putorius furo reference genome (GenBank assembly accession: GCA_000215625.1) (Peng X et al 2014 Nat Biotechnol 32:1250-1255). The alignments were carried out using STAR (v2.7.1a) ( Dobin et al 2013 Bioinformatics 29: 15-21). The ENCODE parameter options for RNA-seq were used (see STAR manual). Gene expression was quantified as gene level counts using the htseq-count function (v0.12.3) (Anders S et al 2015 Bioinformatics 31:166-169).
The Ensembl gene annotations for Mustela putorius furo (genebuild-last-updated 2016-05) was used (Yates AD et al 2020 Nucleic Acids Res 48:D682-D688). The htseq-count default parameters were used except for the strand parameter which was set to reverse to consider the strandedness of library. Differentially expressed genes (DEGs) were identified using DESeq2 (v1.28) (Love MI et al 2014 Genome Biol 15:550). DESeq2 was configured by adding batch and sex to the design to control for these factor whilst DESeq2 tests for association due to the condition (see DESeq2 vignette). Other than including batch and sex in the design, DESeq2 was run with default parameters (Love MI et al 2014 Genome Biol 15:550). Genes were differentially expressed if the absolute log2Fold change was > 1, and the adjusted p-value was < 0.1.
The DESeq2 vs function computes a variance stabilizing transformation of the sample data like putting the data on the log2 scale. To remove shifts in the log2 scale expression data explained by the batch effect, the remove Batch Effect function from limma (v3.44.0) (Ritchie ME et al 2015 Nucleic Acids Res, 43: e4) was used; sex was also included as a factor (see vignettes for DESeq2 and limma).
Genome_build: GenBank assembly accession: GCA_000215625.1) (Peng X et al 2014 Nat Biotechnol 32:1250-1255).
Supplementary_files_format_and_content: excel, DESEQ2, Limma
Submission date Jan 14, 2022
Last update date Oct 19, 2022
Contact name Dr Steven M. Rowe
Organization name University of Alabama at Birmingham
Department Professor and Director, Cystic Fibrosis Research Center
Lab Professor Dr Steven Rowe
Street address 1720 2nd Avenue South, MCLM 804
City Birmingham
State/province AL
ZIP/Postal code 35294
Country USA
Platform ID GPL28369
Series (1)
GSE193749 RNA-Sequencing of COPD ferret lung reveals uniquely expressed gene common to human COPD.
BioSample SAMN25006514
SRA SRX13792636

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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