title: Illumina NovaSeq 6000 paired end sequencing; Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration
design: Transcriptomic and proteomic retinal pigment epithelium signatures of age-related macular degeneration
library_construction_protocol: RPE cells were harvested with 0.25% Trypsin-EDTA for 8 min, inactivated with RPE medium, and dissociated using manual trituration to yield a single-cell suspension. The cell suspension was centrifuged (5 minutes, 300 g, 4 °C), following which cells were resuspended in 1 mL of 0.1% BSA in PBS solution. Subsequently, cells were counted and assessed for viability with Trypan Blue, then pooled (eight samples maximum) at a concentration of 1000 live cells/μl (1*10^6 cells/mL) for single cell RNA-sequencing. Punch biopsies were obtained from subjects over the age of 18 years. Fibroblasts were expanded, cultured and banked in DMEM with high glucose, 10% fetal bovine serum (FBS), L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (all from Thermo Fisher Scientific, USA). All cell lines were mycoplasma-free (MycoAlert mycoplasma detection kit, Lonza, Switzerland). Fibroblasts at passage (p) 2 were used for reprogramming into iPSCs. iPSCs were maintained and passaged using a TECAN liquid handling platform, and cultured onto vitronectin XF™-coated plates (Stem Cell Technologies) in StemFlex™ (Thermo Fisher Scientific), with media changes every second day and weekly passaging using ReLeSR™ (Stem Cell Technologies). Reprogrammed RPE cells were maintained on RPE medium (Alpha-MEM, 5% FBS, non-essential amino acids, N1 supplement, penicillin - streptomycin - glutamate, taurine - hydrocortisone - triiodothyronine) and cultured for a further 22-29 days with media changes every 2-3 days. Cells were then passaged with 0.25% trypsin-EDTA and plated onto growth factor-reduced Matrigel-coated plates (Corning) to enrich in RPE cells for an additional 30 days (76-88 days total). Fibroblasts were reprogrammed into iPSCs through the nucleofection (Lonza) of episomal vectors expressing OCT-4, SOX2, KLF4, L-MYC, LIN28 and shRNA against p53  in feeder- and serum-free conditions using TeSR™-E7™medium (Stem Cell Technologies). Pluripotent cells were selected using anti-human TRA-1-60 Microbeads (Miltenyi). Pluripotency was assessed by expression of the markers OCT3/4 (sc-5279, Santa Cruz Biotechnology) and TRA-1-60 (MA1-023-PE, Thermo Fisher Scientific; ab16288, Abcam) by immunocytochemistry, and virtual karyotyping by CNV array on all lines. iPSCs were differentiated into RPE using E6 medium (Stem Cell Technologies) containing N2 supplement (Life Technologies), penicillin - streptomycin (Life Technologies) for 21-38 days (to reach RPE differentiation). Multi-donor single cell suspensions were loaded on to the wells of 10x Genomics Single A Chip and cDNA was extracted using the Chromium Single Cell 3′ Library & Gel bead kit (10x Genomics; PN-120237). cDNA was amplified through reverse transcription performed using a  C1000 Touch Thermal Cycler with a Deep Well Reaction Module (Bio-Rad) as follows: 45 min at 53 °C; 5 min at 85 °C; hold 4 °C. cDNA was recovered and purified with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific; catalog no. 37002D). Subsequently, it was amplified as follows: for 3 min at 98°C; 12× (for 15 sec at 98 °C; for 20 sec at 67 °C; for 60 sec at 72°C); for 60 sec at 72 °C; hold 4 °C followed recommended cycle number based on targeted cell number. Amplified cDNA was purified with SPRIselect beads (Beckman Coulter; catalog no. B23318) and underwent quality control following manufacturer's instructions.