GSM9065082: Human intestinal organoid cultured in matrigel or type I collagen (hashtag library); Homo sapiens; RNA-Seq
Single-cell suspensions were obtained from Matrigel-embedded organoids on day 7, while collagen-embedded organoids were collected on days 2 and 4 after the start of culture. For Matrigel-embedded organoids, Cell Recovery Solution (Corning, #354253) was used to dissolve the matrix. For collagen-embedded organoids, digestion was performed using collagenase solution (0.5 mg/mL in PBS; Sigma-Aldrich, #C7657). After matrix removal, organoids were incubated in TrypLE Express (Thermo Fisher) for 15–30 minutes. Mechanical dissociation was carried out by pipetting or using a 5 mL syringe to generate single-cell suspensions. The resulting cells were filtered through a 40 μm cell strainer (Falcon, #352340) to eliminate debris and cell aggregates. Isolated cells were stained with biotinylated anti-human CD298 and CD147 antibodies, followed by incubation with TotalSeq PE-conjugated streptavidin (BioLegend). Library construction was conducted by ImmunoGeneTeqs, Inc. Briefy, obtained single-cell suspensions were subjected to a BD Rhapsody system according to the manufacturer's protocol. The reverse-transcribed, exonuclease-I treated BD Rhapsody beads were subjected to TAS-Seq protocol (Shichino et al. Commun Biol. 2022) for cDNA and hashtag library amplification. Whole-transcriptome library was subjected to fragmentation/end-repair/A-tailing using NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs). Pooled libraries were sequenced by Illumina Novaseq 6000 sequencer (Illumina).