title: GSM6792831: donor 3, RPE, macula, atac; Homo sapiens; ATAC-seq
library_construction_protocol: Donor eyes were obtained from the Iowa Lions Eye Bank following full consent of the next of kin and in full compliance with the Declaration of Helsinki. Anterior segments were removed at the limbus and posterior poles were dissected with 4 cuts at 90 degree intervals. Following removal of the vitreous and neural retina, the RPE was gently scraped away from the underlying Bruch's membrane and choroid using a Beaver-Visitec #6900 surgical blade (TestEquity). RPE cells were collected from either the submacular region (corresponding to the central ~8mm) or from the temporal quadrant. Cryopreserved RPE samples were thawed rapidly at 37˚C and washed once in PBS-/-. Cells were then pelleted and nuclei were isolated using the Chromium Nuclei Isolation Kit (10X Genomics) following manufacturer's suggested protocol for Single Cell Multiome ATAC and Gene Expression libraries. Briefly, cells were resuspended in Lysis Buffer on ice and dissociated with a pestle. Dissociated material was filtered with the supplied column and nuclei were subsequently washed once with Wash Buffer. Nuclei were resuspended in diluted Nuclei Buffer supplemented with RNase Inhibitor (10X Genomics). Nuclei were counted using trypan blue exclusion on a Luna automated counter. Nuclei were then processed following the 10X Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression (Rev. E). For each sample, 6,000 nuclei were targeted. Eight cycles were used for ATAC library index PCR. Nine cycles were used for cDNA amplification. Sixteen cycles were used for Gene Expression Library sample index PCR. Library quality was checked using the Bioanalyzer system prior to sequencing. Gene expression and ATAC libraries were sequenced separately on the Novaseq 6000 platform (Illumina). Sequencing was carried out by the Genomics Division of the Iowa Institute of Human Genetics.