Total RNA was isolated from each retina and processed separately using a NucleoSpin RNA Plus XS kit (TaKaRa, Tokyo, Japan), and a library was subsequently prepared using a Switching Mechanism At the 5’ end of RNA Template (SMART) method. Purified amplicon was sequenced using a Novaseq 6000 sequencer (Illumina K.K. San Diego, CA). Raw bead counts were obtained and processed using a web portal for integrated differential expression and pathway analysis (iDEP.95/iDEP1.0; http://bioinformatics.sdstate.edu/, accessed on 24 February 2023) (Ge et al., 2018 (link)). k-Means clustering was performed on the top 2000 genes ranked by standard deviation into groups based on their expression pattern across all samples. A heatmap was subsequently generated in which numerical values of points were represented by a range of colors. Identification of differentially expressed genes (DEGs) was performed using a DESeq2 algorithm by extraction with an FDR cutoff of 0.1 and min-fold change of 2 as the default setting. Pathway analysis was performed using Parametric Gene Set Enrichment Analysis (PGSEA) with gene sets from the Gene Ontology Molecular Function and Pathway and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The cutoff for significance was set at 0.2.
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