title: GSM6131956: Control, periphery, neural retina (scRNA-seq); Homo sapiens; RNA-Seq
library_construction_protocol: Neural retina was dissected away from the underlying retinal pigment epithelium and choroid prior to tissue dissociation. Neural retina samples were dissociated with 20 units/mL of papain (Worthington Biochemical Corporation) with 0.0005% DNase I (Worthington Biochemical Corporation) on a shaker at 37˚C for 75 minutes. RPE/choroid samples were dissociated mechanically using razor blades into one-millimeter pieces then dissociated enzymatically using collagenase on a shaker at 37˚C for 60 minutes. Following dissociation, cells were cryopreserved in DMSO-based Recovery Cell Cryopreservation Media (Gibco). After overnight cooling at -80˚C, samples were transferred to liquid nitrogen (vapor phase) for long-term storage prior to encapsulation and library preparation. Cryopreserved cells were rapidly thawed at 37 °C and resuspended in dPBS-/- (Gibco) with 0.04% non-acetylated bovine serum albumin (New England Biolabs). Cells were filtered through a 70µm filter and diluted to target 8,000 cells per run. Single cells were then partitioned and barcoded with the Chromium Controller instrument (10X Genomics) and Single Cell 3' Reagent (v3.1 chemistry) kit (10X Genomics) according to the manufacturer's specifications with no modification (Rev C). Final libraries were quantified using the Qubit dsDNA HS Assay Kit (Life Technologies) and diluted to 3ng/µL in buffer EB (Qiagen). Library quality and concentration was confirmed using the Bioanalyzer High Sensitivity DNA Assay (Agilent) prior to sequencing.