Computational protocol: Gfi1Cre mice have early onset progressive hearing loss and induce recombination in numerous inner ear non-hair cells

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Protocol publication

[…] RNA was extracted from cochlear ducts of eight day old (P8) Gfi1Cre/+ and Gfi1+/+ mice in independent biological triplicate samples for each genotype using the Direct-zol RNA Mini-prep kit (Zymo Research, Irvine, CA). Each sequenced Gfi1+/+ sample consisted of pooled RNA from two cochleae (1 mouse), and each Gfi1Cre/+ sample consisted of pooled RNA from six cochleae (3 mice). RNA-seq libraries were prepared from a minimum of 75ng of RNA with the TruSeq RNA Sample Prep kit (Illumina) per manufacturer’s protocol, and samples were sequenced on an Illumina HiSeq 2500 with a paired-end 150 base configuration (between 38 and 50 million reads were obtained for each sample, see ). Sequenced reads were aligned to the mouse genome (mm10) using Tophat. Only uniquely mapped reads were used in subsequent analyses. Gene expression levels were calculated using HTseq-count and normalized using quantile normalization. Only genes covered by at least 40 reads were considered as expressed in our dataset (14,866 genes). Differential expression analysis was done using DEseq. RNA-seq data was submitted to the Gene Expression Omnibus database (accession ID GSE85519). Gene expression data from mouse macrophages were taken from GEO RNA-seq datasets GSE65530 and GSE77885. Only unstimulated samples were analyzed. Genes covered by at least 40 reads were called as expressed genes in these datasets. Quantile normalization was applied to allow comparison of expression levels between different datasets. The definition of the set of genes whose expression is enriched in macrophages was based on the comparison of expression between macrophages and sensory epithelial, neuronal, mesenchymal and endothelial cells in the inner ear taken from Hertzano et al.. 360 genes showed at least 3-fold elevated expression in macrophages compared to all other cell types. […]

Pipeline specifications

Software tools TopHat, HTSeq, DESeq
Databases GEO
Application RNA-seq analysis
Organisms Mus musculus