GSM8996618: Organoid Day 3; Homo sapiens; RNA-Seq
Single-cell RNA sequencing (scRNA-seq) of VMOs was performed using the PIPseq pipeline (PIPseq T20 3' Single-Cell RNA kit v4.0, Fluent BioSciences). Organoids collected at D3 and D14 were digested in TrypLE for 20-60 minutes at 37°C with vigorous pipetting every 20 minutes. The resulting cell suspension was filtered through a 40 µm cell strainer (Greiner Bio-One) to remove undigested Matrigel and large cell clumps. The Dead Cell Removal Microbubble Kit (Akadeum Life Sciences) was used to separate viable from dead cells based on buoyancy. Viable cells were then spun down to the required concentration and submitted to core facilities for processing according to the manufacturer's protocol. The PIPSeq T20 kits were used to capture mRNA, synthesize cDNA, and construct libraries. Libraries that passed QC were sequenced in paired-end mode using a 138-cycle NovaSeq X SP kit (Illumina), with read lengths specified by the user guide. The resulting FASTQ files underwent QC preprocessing.