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 Overall design: 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).

用于鉴定斑马鱼视网膜再生过程中小胶质细胞与巨噬细胞转录组的RNA测序（RNA-seq）实验
整体实验设计：
通过向mpeg1:GFP转基因斑马鱼注射终浓度为2 μM的哇巴因，构建可诱导视网膜内层神经元死亡的视网膜损伤模型。本实验共纳入32尾斑马鱼。哇巴因损伤后7天（7 dpi）时，将8个视网膜混合为1份样本，共制备4份生物学样本；并参照Chi等2018年发表于《Experimental Eye Research》（DOI: 10.1016/j.exer.2018.08.002）的方法，从解离的视网膜组织中制备单细胞悬液。随后通过荧光激活细胞分选术（FACS）从每份样本中纯化GFP阳性与GFP阴性细胞。针对#1至#4的每份样本，均通过FACS分离纯化mpeg1:GFP阳性与阴性细胞，提取RNA后，使用Lexogen 3'端mRNA测序制备试剂盒（正向引物型）构建Illumina测序文库。使用Illumina HiSeq 4000测序平台进行SE75单端测序。使用Salmon v0.9.1软件，基于准映射模式并设置"--noLengthCorrection"参数，将测序读段比对并定量至Ensembl数据库第90版的斑马鱼（Danio rerio）转录组（GRCz10）。