GSM5742458: Retina Day 14; Mus musculus; RNA-Seq
Neuroretinal tissues were collected under a dissecting microscope, and the tissues were rinsed three times with PBS. First, the neuroretinas were digested using 0.25% trypsin for approximately 30 min at 37°C until a single-cell suspension was made. Complete DMEM was then used to neutralize the cell suspension. The single-cell samples were passed through a 40 μm cell strainer. Second, CD45-positive cells were obtained by sorting with magnetic beads. A hemocytometer with trypan blue was used to count the cells and ensure that the cell viability was approximately 90%. Finally, single-cell samples were subjected to scRNA-seq with a high-throughput droplet-mediated scRNA-seq platform (10× Genomics Chromium). Each retinal tissue sample was diluted to contain approximately 5,000 CD45-positive cells in the appropriate volume. Subsequently, the single-cell suspension, gel beads and oils were added to the 10× Genomics single-cell A chip. After droplet generation, the samples were transferred into 200 μl PCR tubes, and reverse transcription was performed using a T100 Thermal Cycler (Bio-Rad). After reverse transcription, cDNA was recovered using a recovery agent provided by 10× Genomics. SPRIselect beads were used for clean-up of the cDNA, after which the cDNA was amplified for 10 cycles. The concentration of cDNA was detected with a Qubit2.0 fluorometer (Invitrogen). Single-cell cDNA libraries were prepared according to the Chromium Single Cell 3' Reagent Kit v2 user guide (https://support.10xgenomics.com/single-cell-gene-expression/index/doc/user-guide-chromium-single-cell-3-reagent-kits-user-guide-v2-chemistry).