GSM8679586: D28_eGFPneg_rep1; Homo sapiens; RNA-Seq
The differentiation of hiPSCs into retinal organoids (ROs) was according to published protocols (Li et al., 2019; Li et al., 2018; Luo et al., 2018; Zhong et al., 2014). Briefly, on day 0 (D0) of differentiation, hiPSCs were dissociated to induce embryonic bodies (EBs) formation. EBs were gradually transitioned into neural induction medium (NIM) containing DMEM/F12 (1:1) (GIBCO), 1% N2 supplement (GIBCO), 1% minimum essential media-non essential amino acids (NEAAs, GIBCO), 2 mg/ml heparin (Sigma-Aldrich). On D4 to D6, EBs were seeded onto Matrigel-coated dishes containing NIM, and then switched to retinal differentiation medium (RDM) containing DMEM/F12 (12: 5) supplemented with 2% B27 (without vitamin A, GIBCO), 1% NEAAs and 1% antibiotic-antimycotic (GIBCO) on D16. Since the fourth week of differentiation, optic vesicles (OVs) were gradually formed and morphologically identifiable. OVs were lifted up and cultured in suspension with RDM. The medium was supplemented with 10% fetal bovine serum (FBS, Natocor), 100 mM Taurine (Sigma-Aldrich) and 2 mM GlutaMAX (GIBCO) on D42. Since D90, the medium was supplemented with 1% N2 instead of B27 (without vitamin A). 3D ROs collected at D28, D55, D118, and D170 were dissociated into single cells by Papain Dissociation System (Worthington Biochemical) according to the manufacture' instructions. Cell suspension was centrifuged at 400g for 10 minutes (Allegrax-15R, Beckman Coulter), and cell pellets were resuspended in sorting buffer (PBS containing 1 mM EDTA (GIBCO), 2% [vol/vol] FBS). Cells were sorted at 4℃ by FACSAriafusion (Becton Dickinson). eGFP positive and eGFP negative cells were separately collected in collecting buffer (medium plus 50% [vol/vol] FBS). FSC-A and FSC-H were examined for all cells to obtain live single eGFP positive cells. ROs derived from  parental hiPSCs were used to determine gate for sorting.Total RNA were extracted using Trizol reagent kit (Invitrogen). RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit (Qiagen), end repaired, Abaseadded, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).