title: GSM8242180: Control RO1, D130; Homo sapiens; RNA-Seq
library_construction_protocol: Small aggregates of iPSCs were suspended in ice-cold Matrigel (CorningTM, Cat. 354234). After incubating at 37 °C with 5% CO2 for 20 min, the iPSC/Matrigel clump was gently dispersed in N2B27 medium [DMEM/F12 (GibcoTM, Cat. 11330032): neurobasal medium (GibcoTM, 21103049) = 1:1, 0.5× B27 supplement (GibcoTM, Cat. 21103049), 0.5× N2 supplement (GibcoTM, Cat. 21103049), 0.1 mM β-mercaptoethanol (GibcoTM, Cat. 21985023), and 2 mM GlutaMAX (GibcoTM, Cat. 35050061)] for floating culture in 24-well plates. Around day 14, the adherent cultures were partially lifted at their edges via a 25-min dispase treatment (0.5 mg/mL in DMEM/F12), rinsed with DMEM/F12 medium, and then grown in a B27 medium [DMEM/F12 (3:1), 2% B27, and NEAA (GibcoTM, Cat. 11140050)]. In about 1 week, ROs spontaneously formed, displaying a bright ring when viewed under an inverted microscope, and were grown (three to five ROs per well on low-attachment 24-well plates) in a serum medium [DMEM/F12 (3:1), 8% FCS (GibcoTM, Cat. 26140095), 2% B27, NEAA, 100 mM taurine (Sigma, T0625), and 2 mM GlutaMAX]. Single-cell RNA-Seq was performed at the Single Cell Genomics Core at Baylor College of Medicine. The single-cell cDNA library preparation and sequencing followed the manufacturer's protocols (https://www.10xgenomics.com). Single-cell suspension at 1000 cells/µl in PBS was loaded on a Chromium controller to generate single-cell GEMS (Gel Beads-In-Emulsions). The scRNA-Seq library was prepared with chromium single cell 3' reagent kit v3.1 from 10x Genomics. The product was amplified by PCR and sequenced on Illumina Novaseq 6000 (https://www.illumina.com).