GSM6753950: NR; Homo sapiens; RNA-Seq
Eyeballs from six donors were divided into five tissue regions within 32 hours including the iris, pars plicata, pars plana, RPE, and neuroepithelial layer (Donors 1-6, Table S1). The samples were finely minced with scalpels and dissociated in buffer containing papain (20 U/ml, LS003119, Worthington) supplemented with DNase-I (2000 U/ml, 10104159001, Roche) in Earle's Balanced Salt Solution at 37°C for 30 min. The cell suspensions were resuspended in Earle's Balanced Salt Solution and passed through a 30-µm cell strainer. Then, the viability of the samples was evaluated with AO/PI prior to loading on the single-cell platform. The samples displayed 90% viability on average. Finally, single-cell samples were subjected to scRNA-seq analysis with a high-throughput droplet-mediated scRNA-seq platform (10× Genomics Chromium).  The appropriate volume of each sample was diluted to the appropriate numbers of tissue cells (pars plicata, pars plana, RPE, Iris and NR). Subsequently, the single-cell suspension, gel beads and oils were all added to the 10× Genomics single-cell A chip. After droplet generation, samples were transferred into 200 μl PCR tubes, and reverse transcription was performed using a T100 Thermal Cycler (Bio-Rad, California, USA). After reverse transcription, cDNA was recovered using a recovery agent provided by 10× Genomics. SPRIselect beads were used for cDNA clean-up, and then the cDNA was amplified for 10 cycles. The concentration of cDNA was detected by a Qubit2.0 fluorometer (Invitrogen, Canada). Single-cell cDNA libraries were prepared according to the Chromium Single Cell 3' Reagent Kit v3 user guidance.