Computational protocol: Contrasting Nav1.8 Activity in Scn10a−/− Ventricular Myocytes and the Intact Heart

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Protocol publication

[…] RNA was extracted from 2 freshly harvested mouse myocyte samples, using Qiagen's Rneasy Mini Kit. The kit protocol was followed with the additional step of on‐column DNase digestion.The short‐read sequence files generated by Illumina's CASAVA pipeline were first processed by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Mean base‐call quality scores were above pred‐like values of 32 in all positions of all samples, and no hard trimming of the reads was necessary. The sequence files were then aligned to mouse assembly build mm10 using the Tophat2 splice junction mapper (http://ccb.jhu.edu/software/tophat/index.shtml). Transcripts were assembled by and differential expression analysis was performed with Cufflinks (http://cole-trapnell-lab.github.io/cufflinks/) using the bias‐correction (‐u) and stranded‐library (fr‐firststrand) options. RefSeq gene annotation was incorporated during the “cuffmerge” step to allow for quantification of known as well as novel transcript isoforms. In the final “cuffdiff” step, individual samples were merged into WT and Scn10a −/− replicate groups. […]

Pipeline specifications

Software tools BaseSpace, FastQC, TopHat, Cufflinks
Application RNA-seq analysis
Organisms Mus musculus
Chemicals Sodium