title: GSM4490476: donor_24_mac_CD31_pos; Homo sapiens; RNA-Seq
library_construction_protocol: A 12-mm macular centered punch of RPE/choroid was acquired from four human donors (Donors 8-11). After dissection away from the retina and sclera, the RPE/choroid was finely diced into small squares (approximately 1 mm x 1 mm) with a razor blade. The resulting suspension was dissociated in 450 units/mL of collagenase II (Gibco, Grand Island NY) reconstituted in Hank's Balanced Salt Solution containing calcium chloride and magnesium chloride (Life technologies, Grand Island NY). Dissociated cells were resuspended in DMSO-based Recovery Cell Culture Freezing Media (Life Technologies Corporation, Grand Island NY). Suspensions were placed in a Cryo-Safe cooler (CryoSafe, Summerville SC) to cool at 1°C/minute in a -80°C freezer for 3-8 hours before storage in liquid nitrogen. Cryopreserved cells from single-cell donors 22-25 were rapidly thawed before pre-enrichment for endothelial cells. Cells were incubated for 15 minutes with anti-CD31 microbeads (Miltenyi Biotech, Bergisch Gladbach Germany) according to the manufacturer's instructions before automated autoMACs magnetic separation (Miltenyi Biotech, Bergisch Gladbach Germany) into CD31+ and CD31-cell fractions. Sequencing of both CD31-positive and CD31-negative fractions was performed on pooled libraries using the Illumina NovaSeq platform (San Diego, CA). Single-cell RNA-Seq
attributes.tag: GEO Accession
attributes.value: GSM4490476