GSM5706965: RPE 3; Homo sapiens; RNA-Seq
The RPE tissues were rinsed three times with phosphate-buffered saline (PBS). First, the tissues were digested using 0.25% trypsin for approximately 30 min at 37°C until a single-cell suspension was made, and then complete Dulbecco's Modified Eagle Mediummodified Eagle's medium (DMEM) was used to neutralize the cell suspension. The single-cell samples were passed through a 40 μm cell strainer. Second, the cell suspension was rinsed three times with PBS and centrifuged for 5 min (1,450 rpm, 4°C). Next, a hemocytometer with trypan blue was used to count the number of cells and ensure that the level of cell viability was approximately 90%. 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 contain approximately 5,000 hRPE cells. 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 v2 user guidance (https://support.10xgenomics.com/single-cell-gene-expression/index/doc/user-guide-chromium-single-cell-3-reagent-kits-user-guide-v2-chemistry).