GSM8895865: Donor1, smoker control; Homo sapiens; RNA-Seq
The tenets of the Declaration for Helsinki for research involving human tissue were followed. Human eyes (n=3) were obtained from the Advancing Sight Network (Birmingham, AL). Preliminary experiments were conducted to determine the viability of RPE that was based on death to enucleation (D-E) time. Single cell RNA-seq on normal eyes from two different 86 yr old female donors with D-E of 3.3 and 6 hr with experimentation time of <24 hours and found that the 3.3 hr D-E interval globe exhibited superior viability (>70%), and nearly double the number of genes detected per cell (1301 vs 708 genes/cell). Therefore, globes with D-E times of < 6 hr and death to experimentation of <24 hr. Specifically, human globes from a 79 yr old female nonsmoker without AMD with a death to enucleation (D-E) time of 4.9 hr, a 67 yr old male smoker without AMD with a D-E of 3.8 hr, and a 67 yr old female smoker with early AMD (Minnesota Grade 2) with a D-E of 1.8 hr were immediately preserved on ice for overnight shipping to Hopkins, and dissected no later than 16 hr postmortem. After removing the anterior segment, the fundus was examined both with the retina and with the retina removed and classified using the Minnesota Grading Scale105. A 6 mm sample of the RPE/choroid in the macula was obtained using a 6 mm punch (Integra Lifesciences, Princeton, NJ) that was centered around the fovea. RPE/choroidal cells were dissociated using Papain Dissociation System (Worthington Biochemical, Lakewood, NJ) following the manufacturer's instructions. Dissociated cells were resuspended in ice-cold PBS, 0.04% BSA and 0.5 U/μl of RNAse inhibitors. Cells were then filtered through a 50μm filter and processed for single-cell RNA-sequencing. Cell viability (>90%) was confirmed by negative staining with trypan blue. ScRNA-seq libraries were generated using the Chromium Next GEM single Multiome ATAC+Gene Expression kit protocol (10X Genomics, Inc.). Nuclei resuspended (10-15k) in 0.1X Nuclei Buffer were incubated with a transposition mix containing transposase. Gel Beads in Emulsion (GEMs) were generated by mixing transposed nuclei, barcoded Gel Beads, and partitioning oil on a chromium NEXT GEM Chip controller. After GEM generation, oligonucleotides included Illumina® P5 sequence barcodes for ATAC library and cDNA included an Illumina® TruSeq Read 1 (read 1 sequencing primer), 16 nt 10x Barcode (for GEX), 12 nt unique molecular identifier (UMI) and a 30 nt poly(dT) sequence. Magnetic beads (SILANE) were used to clean up barcoded products from the obtained GEM-RT reaction mixture. ATAC libraries were amplified with 10 PCR cycles and were sequenced on Illumina NovaSeq with 25,000 read pairs per nucleus. RNA libraries were amplified from cDNA with 14 PCR cycles and were sequenced on Illumina NovaSeq with 50,000 reads per nucleus.