GSM9126975: SMG33_T; Homo sapiens; RNA-Seq
Organoids were enzymatically dissociated into single-cell suspensions using TrypLE (Thermo Fisher Scientific) at 37°C for 15 minutes. Cell viability was assessed using acridine orange/propidium iodide (AO/PI) staining and analyzed with a Countstar Fluorescence Cell Analyzer. Cells were then resuspended in PBS or loading buffer and prepared for scRNA-seq library construction. Single-cell RNA-seq libraries were constructed using the Chromium Single Cell 3' v3 Reagent Kits on the 10X Genomics Chromium Controller (10X Genomics, Pleasanton, CA). Approximately 1000 cells/μL were loaded to generate single-cell Gel Bead-In-Emulsions (GEMs). Reverse transcription was performed in each GEM, and barcoded cDNA was purified and amplified. The resulting cDNA was fragmented, end-repaired, A-tailed, and ligated with sequencing adapters, followed by indexed PCR amplification. Final libraries were quantified using the Qubit High Sensitivity DNA Assay (Thermo Fisher Scientific) and sized using the High Sensitivity DNA chip on a Bioanalyzer 2200 (Agilent). Libraries were sequenced on an Illumina platform with 150 bp paired-end reads.