title: RPD_pool_3
organism: Homo sapiens
description: Protocols: RPE cells were dissociated using trypsin-EDTA (0.25%) in phenol red (Life Technologies, 25200072) for 5 min at 37°C in a 5% CO2 incubator, the plate was tapped to release any non-RPE cells and washed with PBS-/- (Life Technologies, 14190-144). Trypsin-EDTA (0.25%) in phenol red (Life Technologies, 25200072) was added a second time and incubated for 10 min at 37°C in a 5% CO2 incubator. The trypsin was inactivated with RPE medium and cells were then harvested into a 15 mL conical tube, as a single-cell suspension. The cell suspension was centrifuged (5 minutes, 300 g, 4°C), then resuspended in 1 mL of 0.1% BSA-PBS. Cells were counted and assessed for viability with trypan blue, and pooled (eight samples maximum) at a concentration of 1000 live cells/μL (1 × 106 cells/mL). The pooled samples were combined with the master mix containing the reverse transcriptase reaction and added to the first well of the third row of the 10x Genomics Chromium Test Chip. Partition oil was loaded into the first well of the first row, and gel beads were added to the first well in the second row. The remaining wells were filled with 50% glycerol and the bottom row was empty. As the chip runs, an emulsion occurs wherein each oil droplet contains one bead and one single cell termed GEM.  Single cell RNA-sequencing libraries were prepped using the Chromium Single Cell 3' Library & Gel bead Kit for 10X Genomics.