title: GSM9065080: Mouse intestinal organoid cultured in matrigel or type I collagen (hashtag library); Mus musculus; RNA-Seq
library_name: GSM9065080
library_construction_protocol: Four days after the start of culture, single-cell suspensions were obtained from each type of organoid. For Matrigel-embedded organoids, Cell Recovery Solution (Corning, #354253) was used, while collagen-embedded organoids were digested with collagenase solution (0.5 mg/mL in PBS; Sigma-Aldrich, #C7657). The collected organoids were then incubated in TrypLE Express (Thermo Fisher) for 15–30 minutes. Mechanical dissociation was performed using pipetting or a 5 mL syringe to obtain single-cell suspensions. The resulting cells were filtered through a 40 μm cell strainer (Falcon, #352340) to remove debris and aggregates. Isolated cells were incubated with biotinylated anti-mouse EpCAM antibodies, followed by staining 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).
identifiers.namespace: GEO
identifiers.id: GSM9065080_r1