GSM8160829: E63_6_GFP-; Sus scrofa; RNA-Seq
Freshly dissected pancreata were minced into fine pieces and digested with collagenase V (0.5 mg/mL for E20-50, 1 mg/mL for E60-85, Sigma-Aldrich) for 5 mins followed by dissociation with TrypLE™ Express (Gibco) for 10-15 mins at 37°C. Cell suspension was filtered through a 40 µm cell strainer, and stained with EpCAM antibody in 1% BSA + PBS (1:200, Invitrogen) for 30 mins at 4°C. Cells were then washed and filtered into a tube through a 35 µm filter. 7-AAD (Invitrogen) was used to distinguish dead cells. The resulting cell suspension was loaded onto a FACSAria III (BD) for sorting. 7-AAD-/EpCAM+ cells from pancreata were collected and pancreata of INS-eGFP embryos at specified ages were used to enrich eGFP+/7-AAD-/EpCAM+ endocrine cells. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, SC-islet 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 Ilumina 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 apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.