Computational protocol: Pias3 is necessary for dorso-ventral patterning and visual response of retinal cones but is not required for rod photoreceptor differentiation

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Protocol publication

[…] Whole transcriptome analysis was performed using three independent biological replicates from retina of Pias3+/+ and Pias3−/− mice at P21 and at 18 months of age. Total RNA was extracted by Trizol LS reagent (Thermo Fisher), according to the manufacturer's protocol, and its quality was assessed using an Agilent 2100 bioanalyzer and RNA 6000 Nano chip. Only RNA samples with RIN≥7.9 were used. Stranded RNA-seq libraries were constructed from 100 ng of total RNA using a modified TruSeq RNA Sample preparation kit protocol (). Paired-end sequencing of 100 bases length was performed on HiSeq 2500 system (Illumina). Pass-filtered reads were mapped using TopHat v2.1.1 () and aligned to UCSC mouse reference genome mm10. Count table of the gene features was obtained using FeatureCounts (). Normalization, differential expression analysis and FPKM values were computed using EdgeR (). An FPKM filtering cutoff of 2 in at least one of the 6 samples was applied. FDR≤0.05 was considered significant and a cutoff of fold change of 1.5 was applied to identify differentially expressed isoforms. R packages and JMP Software (SAS) were used for data mining. GO annotation and pathways enrichment analysis was performed using Panther Classification System ( […]

Pipeline specifications

Software tools TopHat, Subread, edgeR
Application RNA-seq analysis
Organisms Mus musculus, Conus striatus