Fibroblasts from 4 donors with age-related macular degeneration and 3 controls with no history of AMD were grown and treated with modified messenger ribonucleic acid (mRNA) encoding reprogramming factors, Oct3/4, Sox-2, Klf4, and c-Myc, nanog and Lin28. Newly generated iPSC colonies were picked and expanded using Nutristem medium (Stemgent, Reprocell, MD, USA) or MTeSR™1 media. iPSC colonies were detached and grown as embryoid bodies (EB) in EB formation medium and STEMdiff™ Neural Induction Medium and RPE differentiation medium containing DMEM/F12, 2% B-27 supplement, minimum essential media, non-essential amino acids and antibiotics. At ~45 days, pigmented iPSC-derived RPE cells appeared in the cultures. Patches of pigmented iPSC-derived RPE cells were micro-dissected, dissociated with trypsin-ethylenediaminetetraacetic acid (EDTA, 0.05%), and plated onto laminin-coated plates until confluent as previously described.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from differentiated iPSC-derived RPE cells using Aurum™ Total RNA Mini Kit (Bio-Rad Laboratories, CA, USA) according to the manufacturer’s instructions, with Dnase I treatment. RNA quantity and quality were assessed by micro-volume spectrophotometry on the Nanodrop 2000 (Thermo Fisher Scientific) and by on-chip capillary electrophoresis on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).
Label
biotin
Label protocol
GeneChip® WT Plus Reagent Kit were used for generating biotinylated ss-cDNA for hybridization.
Hybridization protocol
All reactions and hybridizations were carried out according to the manufacturer's protocol (Affymetrix; Thermo Fisher Scientific). Affymetrix GeneChip Human Clariom S arrays (Thermo Fisher Scientific) were washed using the GeneChip® Fludics Station 450 (Thermo Fisher Scientific)
Scan protocol
Affymetrix GeneChip Human Clariom S arrays were scanned with the GeneChip Scanner 3000 (Thermo Fisher Scientific).
Description
RPE AMD 2-1 Avg (log2)
Data processing
Raw data were processed to perform gene-level normalization and quality control using Affymetrix Expression Console Software (Thermo Fisher Scientific). RMA expression value derived from Expression Console software Transcriptome Analysis Console (TAC) 4.0.1 (Applied Biosystems) probe group file: Clariom_S_Human.r1.pgf meta-probeset file: Clariom_S_Human.r1.na36.hg38.a1.transcript.csv Normal vs AMD, Normal: 3 samples, AMD: 4 samples; Filter criteria: Fold Change: > 2 or < -2; Total number of genes: 15606
