Integration of eQTL and single-cell transcriptomics in the human eye identifies causal genes for age-related macular degeneration

Bulk RNAseq of retina and RPE/choroid tissue, each from the macula or non-macula (peripheral) regions of human donor eyes. **Raw Data not provided due to patient privacy concerns** Postmortem eyes from 129 human donors for bulk RNA seq and 5 donors for single-nucleus RNAseq (see below) were procured by the Florida Lions Eye Bank (Tampa, FL). Donors profiled in this study were 43 females and 76 males, ranging from 59 to 98 years of age. From these, 99 donors had no history of ocular diseases and 23 donors were previously diagnosed with AMD. Eyes were enucleated 4 h postmortem or less and preserved in RNAlater immediately after collection. The macula is fully contained within the boundaries of superior and inferior temporal vascular arcades and is easily visualized. After the macula was dissected out from the peripheral fundus using dissecting scissors, the macular retina was separated from the RPE and choroid underneath the retina. Total RNA was isolated from tissues using RNeasy Mini kit (QIAGEN) including the on-column DNase digestion. Quality control of samples was done to determine RNA quantity and quality prior to their processing by RNA-seq. The concentration of RNA samples was determined using NanoDrop 8000 (Thermo Scientific) and the integrity of RNA was determined by Fragment Analyzer (Advanced Analytical Technologies). Samples with RNA integrity number (RIN) below 8 were excluded from further analysis. 0.5 ug of total RNA was used as an input material for library preparation using TruSeq RNA Sample Preparation Kit v2 (Illumina). Size of the libraries was confirmed using 2200 TapeStation and High Sensitivity D1000 screen tape (Agilent Technologies) and their concentration was determined by qPCR-based method using Library quantification kit (KAPA). The libraries were multiplexed and then sequenced on Illumina HiSeq2500 (Illumina) to generate 30M of single end 50 base pair reads. Sequencing data analysis was performed as previously described (Durinck et al., 2015). Sequencing reads were mapped to the reference human genome (GRCh38), using the GSNAP short read aligner (Wu and Nacu, 2010). Expression was quantified using HTSeqGenie as reads per kilobase of gene model per million total reads (RPKM), and normalized using size factors computed using DESeq2 (nRPKM).