RNA Purification, miRNA, and mRNA Profiling

For miRNA profiling, the sorts of 44 light damaged retinas were pooled for RNA purification. RNA was extracted and purified with a miRNeasy Micro Kit in accordance with manufacturer's instructions (Qiagen). NanoString nCounter was used for miRNA expression analysis. Two hundred ng total RNA per sample (33 ng/μl) was submitted for NanoString analysis. NanoString data was analyzed using nSolver 4.0 software. The data represents counts of molecules normalized against 4 housekeeping genes (β-actin, GAPDH, Rpl19, and B2m), 8 negative controls, and 6 positive controls that were run with the samples. miRNA data after Dicer-cKO and for wild type MG was published before (Wohl et al., 2017), is available at GEO (GSE 103098). Raw data was de-novo normalized and analyzed together with the light damaged data. For Microarray, 12 retinas were used for controls and 10 retinas for light damage, FACS-purified, the RNA isolated and run on the Mouse Gene 1.0 ST microarray (Affymetrix) according to manufacturer's guidelines. The RIN numbers for the samples ranged from 8 to 10 with a mean of 8.98. The microarray data was normalized and analyzed with Affymetrix Power Tools software and TM4 Multi-Experiment Viewer software. RNA-Seq data of pigmented adult wild type MG was published before (Wohl et al., 2017), is available at SRA (NBCI, SRP115835) and was used for gene expression comparisons. Also, the datasets of control FACS purified MG and MG 36h after light damage, as well as 48h after NMDA damage from Hoang et al. (2020) were used for gene expression comparisons.