biostudies-arrayexpressJessica Yuen Wuen MaHomo sapiensCell Rangerhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16297This study investigates the differentiation of retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) to optimise and characterise in-vitro RGC generation. Retinal organoids were derived from a human embryonic stem cell line (H9) and a laboratory-established human induced pluripotent stem cell (hiPSC) line. For each stem cell line, two independent passages of cells were generated as biological replicates (n = 2 per line). To assess transcriptional diversity and differentiation efficiency, organoids were harvested at day 40 of differentiation, dissociated into single cells, and cultured for an additional 14 days to enrich for RGCs. Following enrichment, single-cell suspensions were processed using the 10x Genomics Chromium platform and sequenced on an Illumina NovaSeq X Plus. The resulting single-cell transcriptomes provide a molecular snapshot of RGC differentiation from hPSCs and enable comparison between hESC- and hiPSC-derived retinal lineages. The data contribute to refining differentiation protocols and improving reproducibility of stem-cell-derived retinal models. Raw BCL files were processed at the Australian Genome Research Facility (AGRF) using Illumina’s standard pipeline to generate FASTQ files. FASTQs were additionally processed with Cell Ranger v7.1.0 (10x Genomics) using the GRCh38/Ensembl v109 reference to produce unfiltered gene-by-cell UMI count matrices in standard 10x sparse format (barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz), which are provided as processed data in this submission. Raw FASTQ files are also archived in this ArrayExpress submission.biostudies-arrayexpressSample Collection - Human pluripotent stem cell (hPSC)-derived retinal organoids were generated from a human embryonic stem cell line (H9) and a laboratory-established human induced pluripotent stem cell line (WAB-00222). Organoids were harvested at differentiation day 40 and dissociated using the Papain Dissociation Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Briefly, tissue was incubated in papain/DNase solution at 37 °C for 20 minutes, gently triturated, and the reaction was terminated using the albumin-ovomucoid inhibitor solution. Cells were washed in 1 % bovine serum albumin (BSA), filtered sequentially through 30 µm preseparation filters (Miltenyi Biotec, 130-041-407), and maintained on ice. Cell viability and concentration were assessed using Trypan Blue exclusion on a Countess 3 FL Automated Cell Counter (Thermo Fisher Scientific; AMQAF2000). Fixed cell suspensions were then prepared using the GEM-X Flex Sample Preparation v2 Kit (10x Genomics; 1000781) following protocol CG000782 for single-cell gene expression.Sequencing - Indexed single-cell gene expression libraries were sequenced at the Australian Genome Research Facility (AGRF) on an Illumina NovaSeq X Plus using a 10B flow cell and paired-end 150 bp read configuration. Base calling was performed using Illumina’s standard software. Raw BCL files were converted to FASTQ format using the standard Illumina pipeline for downstream processing.Nucleic Acid Extraction - The 10x Genomics GEM-X Flex Gene Expression Human 4-plex assay was used for single-cell RNA profiling. Fixed cells were thawed, washed, and counted. Approximately 300,000 cells from each biological sample were used for probe hybridisation. Cells were resuspended in a hybridisation buffer containing uniquely barcoded probe sets (BC001-BC004) and incubated for 20 hours at 42 °C to capture RNA molecules in situ. After hybridisation, cells from each sample were recounted, pooled, and washed. The concentration of the pooled suspension was then determined. No separate RNA extraction step was performed, as the assay captures RNA directly within fixed cells.Library Construction - Gel Beads-in-Emulsion (GEMs) were generated using the Chromium X platform. Barcoded Gel Beads, pooled hybridised cells, and Partitioning Oil B were loaded onto the GEM-X FX Chip to target recovery of ~80,000 cells. GEMs were thermocycled to ligate left-hand and right-hand probes hybridised to target RNA, hybridise the Gel Bead primer, and extend barcode sequences. Following ligation and barcode extension, GEMs were broken with Recovery Reagent. A PCR master mix was added to pre-amplify ligated and extended products. Amplified products were cleaned and subjected to sample-index PCR to incorporate Illumina adapters and indexes. Final libraries were purified, quality-checked using an Agilent TapeStation D1000, quantified by qPCR, and sequenced on an Illumina NovaSeq X Plus (10B flow cell; paired-end 150 bp reads with 10 % PhiX spike-in).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Cell Ranger v7.1.0 (10x Genomics) was used to generate unfiltered gene-by-cell count matrices in 10x sparse matrix format (barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz), which are provided as processed data in this submission.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsChromium X instrument (10x Genomics); GEM-X Flex Sample Preparation v2 Kit (10x Genomics; 1000781); Human Gene Expression Probe Set v1.0.1 (10x Genomics)Illumina NovaSeq XChromium X instrument (10x Genomics); GEM-X FX Chips (10x Genomics); Agilent TapeStation D1000; qPCR (library quantification kit for Illumina platforms)NoneCountess 3 FL Automated Cell Counter (Thermo Fisher Scientific; AMQAF2000); GEM-X Flex Sample Preparation v2 Kit (10x Genomics; 1000781)RNA-seq of coding RNA from single cellsHomo sapiensJessica Yuen Wuen MafalseSingle-cell RNA sequencing of human pluripotent stem cell-derived RGC-enriched retinal cells generated from hESC and hiPSC retinal organoidsThis study investigates the differentiation of retinal ganglion cells (RGCs) from human pluripotent stem cells (hPSCs) to optimise and characterise in-vitro RGC generation. Retinal organoids were derived from a human embryonic stem cell line (H9) and a laboratory-established human induced pluripotent stem cell (hiPSC) line. For each stem cell line, two independent passages of cells were generated as biological replicates (n = 2 per line). To assess transcriptional diversity and differentiation efficiency, organoids were harvested at day 40 of differentiation, dissociated into single cells, and cultured for an additional 14 days to enrich for RGCs. Following enrichment, single-cell suspensions were processed using the 10x Genomics Chromium platform and sequenced on an Illumina NovaSeq X Plus. The resulting single-cell transcriptomes provide a molecular snapshot of RGC differentiation from hPSCs and enable comparison between hESC- and hiPSC-derived retinal lineages. The data contribute to refining differentiation protocols and improving reproducibility of stem-cell-derived retinal models. Raw BCL files were processed at the Australian Genome Research Facility (AGRF) using Illumina’s standard pipeline to generate FASTQ files. FASTQs were additionally processed with Cell Ranger v7.1.0 (10x Genomics) using the GRCh38/Ensembl v109 reference to produce unfiltered gene-by-cell UMI count matrices in standard 10x sparse format (barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz), which are provided as processed data in this submission. Raw FASTQ files are also archived in this ArrayExpress submission.2026-05-30T00:00:00Z2026-05-30T01:01:35.911Z2025-12-01T22:36:16.632ZE-MTAB-16297ERP185917EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184