title: GSM8439527: WT_1mon; Mus musculus; RNA-Seq
library_construction_protocol: RPE-choroid tissues were harvested from WT and Vldlr-/- mice at the ages of 1 month and 2 months. The isolation of viable single cells from RPE-choroid tissue was performed according to a published protocol . Briefly, the eyeballs were enucleated and kept in pre-chilled PBS. Extraocular muscles were cleaned from the eyeballs, and a circumferential incision around the limbus was performed to remove the anterior segments. The neural retina was then gently separated from the RPE. RPE-choroid tissues were gently scraped from the sclera and sequentially digested with collagenase A (6.25 mg/ml) and Dispase II (6.25 mg/ml) at 37°C for 15 min and then with Trypsin (0.25%) at 37°C for 5 min. After neutralization in a buffer containing 10% FBS, the tissue suspension was filtered through a 40-μm strainer to generate a single cell suspension. Dead Cell Removal Kit (Miltenyi Biotec, San Jose, CA) was used to eliminate dead cells. A low-speed (200 g, 10 min) and large-volume (40 ml in 50 ml tube) wash was performed to remove the cellular debris. Finally, the dissociated RPE/choroid cells with viability higher than 90%, as measured by a trypan blue assay, were used for cDNA library construction. Cell suspensions in PBS containing 0.5% BSA with a density of approximate 200 cells/μl were used on the 10x Genomics Chromium platform to generate barcoded single-cell gel bead-in-emulsion (GEMs), targeting ~ 5,000 cells per library. Barcoded libraries were sequenced on an Illumina HiSeq4000 (Illumina, USA) at Wake Forest University Cancer Genomics Shared Resource (CGSR).