GSM6380307: D0 and D8, scRNAseq; Homo sapiens; RNA-Seq
For dissociation of organoids into single cells for staining, these organoids were incubated in 50 microliters 2.5% trypsin (Corning) supplemented with 10 mM EDTA (Sigma-Aldrich) for 2 minutes. Well contents were transferred to 1.7 mL tubes and 500 microliters PBS was added to tubes and centrifuged at 300 x g for 4 minutes. Supernatant was aspirated and 50 microliters of Collagenase 4 (STEMCELL Technologies) was added prior to suspension being mechanically agitated by 15 strokes with a P-1000 and 15 strokes with a P-200. Suspension was incubated for 10 minutes prior to mechanical agitation by 30 strokes with a P-200. 750 microliters PBS was added prior to centrifugation at 300 x g for 4 minutes. Supernatant was aspirated and cells were resuspended in 50 microliters before being passed through a 40-micron strainer into a 2.0 mL tube. Cells were washed with 200 microliter PBS and centrifuged at 300 x g for 4 minutes with supernatant discarded before counting. Library was performed according to the manufacturer's instructions (single cell 3' v2 protocol, 10x Genomics) by MIT BioMicroCenter Core. Cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.