Mouse retina single-cell RNA-seq of WT and two Nr2e3 mutant models to explore the role of  Nr2e3 in cone and rod photoreceptor differentiation
Mouse retina single-cell RNA-seq of WT and two Nr2e3 mutant models to explore the role of  Nr2e3 in cone and rod photoreceptor differentiation
Two retinas per mouse were dissected and an equal number of male and female mice were used for each sample. Retinas were dissected in Neurobasal media and placed on ice. Afterwards they were incubated at 37C for 15 min in papain solution with agitation every 5 min. DNase I (10U/ul, Roche) was added and incubated at 37C for additional 5 min. Samples were homogenized by gently pipetting, and cells were centrifuged at 400 g for 5 min at 4C. Methanol fixation and rehydration were performed according to the Single Cell RNA Sequencing guidelines of 10X Genomics. Briefly, a minimum of 1x10^6 cells were resuspended in 200 ul of cold PBS and 800 ul of cold methanol. Cell suspension was placed on ice for 30 min prior to transferring to -80C for long-term storage for up to 6 weeks. For rehydration, cells were placed on ice for 5 min to equilibrate them to 4C. Cells were centrifuged at 1000 g for 5 min at 4C and resuspended in 300 ul of Wash Resuspension Buffer (0.04% BSA, 1mM DTT, 0.2 U/ul RNase inhibitor in 3X SSC buffer). The cell suspension was filtered through a 50-μm cell strainer. Processing for single-cell capture and library preparation was performed according to the 10X Genomics standard protocols. We performed scRNA-seq using the 10X Genomics Chromium Single Cell 3' Reagents v3.1 (10X Genomics PN-1000092), according to the manufacturer's instructions (Rev D). We performed scRNA-seq using the 10X Genomics Chromium Single Cell 3' Reagents v3.1 (10X Genomics PN-1000092), according to the manufacturer's instructions (Rev D).