In order to more closely examine the transcriptome of choroidal endothelial cells, we modified our dissociation protocol prior to a second single-cell sequencing experiment to shift the distribution of analyzed cells to include more endothelial cells. In this experiment, separate 12-mm punches of RPE/choroid were centered on the macula and on the periphery (∼12 mm inferotemporal to the foveal center). Four human donors were included in this study (Table 1 ). Donors 5 to 7 had no documented ophthalmic history, while donor 4 had previously been diagnosed with neovascular AMD. After dissection away from the retina and sclera, the RPE/choroid was finely diced into small squares (∼1 × 1 mm) with a razor blade. The resulting suspension was dissociated in 450 units/mL collagenase II (Gibco) reconstituted in Hank’s balanced salt solution containing calcium chloride and magnesium chloride (Life Technologies) following a previously described protocol (65 (link)). RPE/choroid and collagenase II were incubated on a shaker at 37 °C for 1 h before cryopreservation, as described above.
Cryopreserved cells from donors 4 to 7 were rapidly thawed before preenrichment for endothelial cells. Cells were incubated for 15 min with anti-CD31 microbeads (Miltenyi Biotech) according to the manufacturer’s instructions before automated autoMACs magnetic separation (Miltenyi Biotech) of the CD31+ cell fraction. The positive cell fraction was passed 2 times through a 40-μm filter to remove aggregates. Single cells were immediately barcoded before library sequencing as described above. Cells with unique gene counts fewer than 500 were eliminated, and cells with more than 7,000 unique genes per cell were removed to eliminate potential doublets. Data were log-normalized before canonical correlation analysis aggregation was performed in Seurat.
Cryopreserved cells from donors 4 to 7 were rapidly thawed before preenrichment for endothelial cells. Cells were incubated for 15 min with anti-CD31 microbeads (Miltenyi Biotech) according to the manufacturer’s instructions before automated autoMACs magnetic separation (Miltenyi Biotech) of the CD31+ cell fraction. The positive cell fraction was passed 2 times through a 40-μm filter to remove aggregates. Single cells were immediately barcoded before library sequencing as described above. Cells with unique gene counts fewer than 500 were eliminated, and cells with more than 7,000 unique genes per cell were removed to eliminate potential doublets. Data were log-normalized before canonical correlation analysis aggregation was performed in Seurat.
