Figure 1.

Schematic representation of the Müller glia RNA-seq experimental design. (A) Photoreceptors were ablated in free-swimming Tg(gfap:EGFP)mi2002 fish using an acute light-lesion paradigm. (B) Retinas were dissected from unlesioned controls (0 hpl) and 8 and 16 hpl Tg(gfap:EGFP)mi2002 fish. Retinas were also dissected from unlesioned, wild-type (nontransgenic, GFP−) control fish. (C) Dissected retinas from each group were pooled and dissociated. (D) Dissociated samples were subjected to fluorescence-activated cell sorting (FACS), and GFP+ cells were collected. The FACS plots shown are representative images from the final gating and collection of actual samples. (E) RNA was isolated from the sorted cells and checked by Bioanalyzer for quality and concentration. Samples with an RNA integrity number (RIN) above 7.0 were advanced to library preparation. The Bioanalyzer electropherogram shown is a representative plot from an actual sample with a RIN of 8.6. The x-axis is in seconds, which corresponds to size. The y-axis shows fluorescent units (FU), corresponding to the amount of RNA. (F) RNA-seq libraries were prepared and checked again via Bioanalyzer. The Bioanalyzer electropherogram shown is a representative plot from a sample library preparation. The x-axis shows the size in base pairs. The y-axis shows fluorescent units, corresponding to the amount of DNA. (G) The RNA-seq libraries were then sequenced on an Illumina GAIIx. (H) The sequencing data were processed with bioinformatic tools for differential expression analysis, gene ontology analysis, and pathway analysis.