GSM7785358: 10x_Retina0426Cd73N; Mus musculus; RNA-Seq
We have generated 16 scRNA-seq samples of the mouse C57BL6/J retina. After dissection, retinas were dissociated into single cells using papain-based enzyme following the published protocol 10. With activated 45U of papain (Worthington, Cat. #LS003126) solution (1mg L-Cystine, Sigma; 8 KU of DNase I, Affymetrix; in 5 ml DPBS), retina was incubated at 37C for ~20min. Which was followed by the replacement of incubation buffer with 2ml ovomucoid solution (15 mg ovomucoid, Worthington Biochemical; 15 mg BSA Thermo Fisher Scientific; in 10 ml DPBS) and 500ul deactivated FBS. The retina tissue was carefully triturated and filtered by 20 um plastic mesh. Repeat the step with additional 1ml ovomucoid solution until no tissue is visible. Spin the cells down at 300g, 4C for 10 min, and cells were used in the next step. To deplete the photoreceptors, cells were resuspended in 0.5% BSA and stained with CD73-PE antibody for 10min at 4C (for each million cells, add 98ul 0.5% BSA with 2ul CD73-PE antibody), and washed with 35 ml 0.5% BSA at 4C for 10min. After being stained with Anti-PE microbeads (for each million cells, add 80ul 0.5% BSA and 20ul microbeads), for 15 min at 4C, cells were washed and resuspended in 0.5% BSA. CD73 negative neuronal cells were enriched by autoMACS Pro Separator (Miltenyi Biotec) DEPLETES mode and ready for the next step. Similarly, CD90.2 positive neuronal cells were enriched with CD90.2 microbeads (for each 10million cells, 90ul 0.5% BSA and 10ul CD90.2 microbeads were used) and autoMACS POSSEL-S mode. Cells viability was 85%-87% when checked with DAPI staining under microscope. Guided by 10X manufacturer's protocols (https://www.10xgenomics.com), single cell cDNA library was prepared and sequenced. Briefly, single cell suspension was loaded on a Chromium controller to obtain single cell GEMS (Gel Beads-In-Emulsions) for the reaction. The library was prepared with Chromium Next GEM single cell 3' kit V2 (10X Genomics) and sequenced on Illumina Novaseq 6000 (https://www.illumina.com). Our in-house generated single cell data was sequenced at the Single Cell Genomics Core at Baylor College of Medicine.