GSM8160848: E63_mG_low [RNA-Seq]; 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. GFP+/7-AAD-/EpCAM+ pancreatic epithelial cells were enriched. For nuclei isolation and library construction, a low input nuclei isolation protocol adapted from 10X Genomics was performed. In brief, sorted cells were washed once with 1 mL PBS + 1% BSA, counted, centrifuged, and supernatant was aspirated. Subsequently, the washed cell pellet was resuspended in chilled lysis buffer with 0.5x detergent concentration (50 μL per sample) and placed on ice for 5 min. Then wash buffer (500 μL per sample) was added and nuclei were centrifuged. To gradually change from wash to diluted nuclei buffer, cells were washed once in a 1:1 mixture of wash buffer and diluted nuclei buffer and subsequently one with pure diluted nuclei buffer. The washed isolated nuclei were then resuspended in 7-10 μl diluted nuclei buffer and were directly added to the transposition reaction after quality control and counting. In all following steps, 10X Genomics' Single Cell Multiome ATAC and gene-expression protocols were followed according to the manufacturer's specifications and guidelines.