Computational protocol: MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis

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Protocol publication

[…] Indexed cDNA libraries were prepared using the Ovation Single Cell RNAseq system (Nugen). The multiplexed libraries were loaded and sequences were produced using a TruSeq PE cluster and SBS-kit on a HiSeq 1500 (Illumina). Approximately 25 million paired-end reads/sample were mapped against the mouse reference genome (NCBI Build 37/UCSC mm9) using STAR software to generate read alignments for each sample. Expression levels were quantified using the featureCounts [] tool and the UCSC RefSeq gene annotation as a reference (exons only, genes as meta features). Differential analysis between the groups was performed using the EdgeR package (quasi-likelihood F-tests). Normalized expression levels were estimated using the EdgeR rpm function and converted to log2 FPKM (fragments per kilobase of exon per million mapped reads) after resetting low FPKMs to 1. To perform blind clustering analysis, genes were selected based on the overall variance between samples (independently of their category), by keeping only the 30 most variant ones. Functional analysis was performed using the DAVID web-based functional annotation tool []. […]

Pipeline specifications

Software tools STAR, Subread, edgeR, DAVID
Application RNA-seq analysis
Organisms Mus musculus
Diseases Autoimmune Diseases, Uveitis