GSM9049878: Retinal organoids at day 170 of differentiation, 12h light (constant light) and 12h dark for 7 days, bulk seq 1; Homo sapiens; RNA-Seq
Bulk RNA sequencing: Retinal organoids were washed twice with PBS and dissociated in RNA Blue Reagent (Top-Bio). Samples were either stored at −80 °C or processed immediately. Organoids were homogenized using an insulin syringe, and total RNA was extracted using the Direct-zol™ RNA MicroPrep Kit (ZYMO RESEARCH, Cat. No.: R2062), following the manufacturer's protocol.  Single-cell RNA sequencing: Organoid dissociation was carried out using the Neurosphere Dissociation Kit (#130-095-943, Miltenyi Biotec) according to the manufacturer's instructions. Briefly, organoids were washed twice with cold HBSS (#14025092, Gibco) and transferred into Eppendorf tubes containing 960 μL of preheated Buffer X (each set of 15 organoids was dissociated in a separate tube). Then, 25 μL of Enzyme P, 10 μL of Buffer Y, and 5 μL of Enzyme A were added and mixed gently. The tubes were placed on a thermal mixer with a block (Eppendorf) for 10 minutes at 37°C and 800 rpm. The organoids were dissociated using a fire-polished Pasteur pipette by pipetting up and down 10 times slowly. They were then incubated again under the same conditions for 5 minutes in the thermal mixer and pipetted up and down 10 times with the Pasteur pipette. After adding cold HBSS containing 5% FBS to the single-cell suspension, the mixture was passed through a 40 μm cell strainer placed on a 15 mL tube. Cells were counted, and the tubes were centrifuged at 300×g for 10 minutes at room temperature. The supernatant was aspirated completely, and the cells were resuspended in cold HBSS containing 5% FBS at a concentration of 1000 cells/μL and kept on ice. (Total single cell number 1.500.000/sample) Bulk RNA sequencing: RNA integrity was checked on the Fragment Analyzer using RNA Kit 15 nt (Agilent Technologies). 500ng of total RNA was used as input for library preparation using QuantSeq 3′ mRNA-Seq FWD with UDI 12 nt Kit (v.2) (Lexogen) in combination with UMI Second Strand Synthesis Module for QuantSeq FWD. Quality control for library quantity and size distribution was done using QuantiFluor dsDNA System (Promega) and High Sensitivity NGS Fragment Analysis Kit (Agilent Technologies). Final library pool was sequenced on NextSeq using High Output Kit v2.5 75 cycles (Illumina) in single-end mode.  Single-cell RNA sequencing: Library preparation was conducted using the same protocol. The quality and quantity of the prepared libraries were assessed using the High Sensitivity NGS Fragment Analysis Kit (Agilent Technologies) and the QuantiFluor dsDNA System (Promega). The libraries were then sequenced using the AVITI sequencing platform (Element Biosciences), employing the AVITI 2×75 Sequencing Kit Cloudbreak FS High Output (150 Gb). Sequencing reads were processed using Cell Ranger software version 8.0.1 (10x Genomics), with FASTQ files aligned to the human reference genome refdata-gex-GRCh38-2020-A, also provided by 10x Genomics, to generate gene expression matrices for downstream analyses.