title: GSM6132185: MELAS, periphery, rpechor (scATAC-seq); Homo sapiens; ATAC-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. Cells were prepared for mt-scATAC sequencing using the protocol described previously by Lareau, Ludwig et al. Briefly, cells were thawed as above and filtered through a 70µm cell strainer. Cells were then washed twice with dPBS-/- (Gibco). Cells were fixed with 1% formaldehyde (Sigma) in PBS-/- for 10 minutes at room temperature. Fixation was quenched with the addition of glycine (Research Products International) to a final concentration of 0.125M for 5 minutes at room temperature. Following fixation, cells were washed twice with ice-cold PBS-/-. Following the second wash, cells were resuspended in 100µL ice-cold Lysis Buffer (10 mM Tris-HCl pH 7.4 (Millipore-Sigma), 10 mM NaCl (Ambion/Invitrogen), 3 mM MgCl2 (Ambion/Invitrogen), 0.1% Nonidet-P40 substitute (Research Products International), 1% Fraction V BSA (Research Products International)) and incubated on ice for 2.5 minutes. Following incubation, 1mL of ice-cold Wash Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA) was added, and cells were pelleted at 500 x g for 5 minutes at 4˚C. Cells were resuspended in 10µL diluted Nuclei Buffer (10X Genomics) and counted using Trypan Blue (Gibco) with the Countess automated cell counter (Invitrogen). Based on these counts, cell suspensions were diluted in diluted Nuclei Buffer to a final target concentration of 6,000 cells per µL. Cells were then processed following the 10X Chromium Next GEM Single Cell ATAC Reagent Kit v1.1 User Guide (Rev. D) without modification. Final libraries were quantified using the Qubit dsDNA HS Assay Kit (Life Technologies) and diluted to 3ng/µL. Library quality was checked using the Bioanalyzer instrument (Agilent), wherein periodicity of fragment length was observed, consistent with high quality ATAC-seq library construction.