title: GSM6048031: hiPSC-1; Homo sapiens; RNA-Seq
library_construction_protocol: MGCs were isolated from ROs at D150 after initiation of RO induction. NR layers were isolated from ROs at different timepoints (D60, D90, D120 and D150) after differentiation, dissected into small pieces with a pair of tungsten needles, and then dissociated into single cells by incubation of accutase (Gibco) for 15–25 minutes at 37°C. Afterwards, cell suspension was plated at a density of 1×104 cells per cm2 on Matrigel-coated plates with MGC culture medium consisting of DMEM, 10% FBS, 2 mM GlutaMAX, 1% NEAA and 1% antibiotic-antimycotic. These cells were noted as passage 0 (P0). After cells were expanded, they were passaged at approximately 90% confluence every 6–10 days. Medium was changed every 3–4 days. Samples from BC1-GFP hiPSCs and their derivatives were used to perform RNA-seq and data analysis. HiPSCs (approximate 2×106 cells per experiment, 3 independent experiments), NR layers isolated from D70-ROs and D150-ROs (5 ROs per experiment, 3 independent experiments), P1 and P5 iMGCs from NR layers of D150-ROs (approximate1×106 cells per experiment, 3 independent experiments) were collected in Trizol reagent (Invitrogen) and stored in a -80℃ freezer. Total RNA was extracted using Trizol reagent kit (Invitrogen) according to the manufacturer's protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies).Oligo (dT) beads were used to isolate the poly mRNA from the total RNA. The enriched mRNA was fragmented and reverse transcribed into cDNA using random primers. After synthesis of the second strand, the cDNA was purified, end-repaired and ligated to Illumina sequencing adapters. The ligation products were size selected, amplified, and sequenced.