3' mRNA QuantSeq of microglia/macrophages during zebrafish retinal regeneration following ouabain-induced inner retinal lesion

RNA-seq to identify transcriptome of microglia and macrophages during zebrafish retinal regeneration A retinal lesion causing death of inner retinal neurons was induced by injection of 2μm final concentration of ouabain using mpeg1:GFP transgenic fish. 32 fish total were included in the experiment. At 7 days post ouabain lesion (7 dpi), 4 biological samples were generated by pooling 8 retinas to create one sample, and single cell suspension was prepared from dissociated retinas as detailed in Chi et al. 2018 Experimental Eye Research DOI 10.1016/j.exer.2018.08.002. FACS sorting was then used to purify GFP+ and GFP- cells from each sample. For each sample (#1-4), mpeg1:GFP+ and GFP- cells were isolated/purified by FACS, RNA was extracted,and libraries were made using Lexogen 3' mRNA-Seq Prep Kit FWD for Illumina. Sequencing was performed on a SE75 run using an Illumina HiSeq 4000 instrument. Reads were quantified/mapped using Salmon v0.9.1 using the quasi-mapping-based mode and the "--noLengthCorrection" setting against Ensembl release-90 Danio rerio transcriptome (GRCz10).