title: Single-cell RNA sequencing of human pluripotent stem cell-derived RGC-enriched retinal cells generated from hESC and hiPSC retinal organoids
design: Single-cell RNA sequencing of human pluripotent stem cell-derived RGC-enriched retinal cells generated from hESC and hiPSC retinal organoids
library_construction_protocol: 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.  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.  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). 