GSM7739813
Pancreatic tissue was perfused with 0.5mg/mL collagenase P (Roche, 11213873001) dissolved in 1×HBSS buffer containing 10mM HEPES (pH 7.4), 1mM MgCl2 and 5mM glucose. Digestion was terminated by adding cold PBS. Islets were manually picked up and dissociated into single cell suspension with 0.25% trypsin (Thermo Fisher Scientific, 25200114) and incubated for 3-5min at 37℃. Fetal bovine serum (FBS) was used to terminate the digestion (40% vol/vol). After centrifugation at 1000rpm for 5min at 4℃, the supernatant was removed and the cell pellet was resuspended with HBSS buffer. Cells were directly sorted using a BD Aria SORP flow cytometer. Smart-seq3xpress scRNA-seq was performed following a published procedure. Briefly, single cells were sorted into 384-well plates containing 0.3μL lysis. The mRNA was reverse transcribed into cDNA using 0.1μL dispensed RT mix. The libraries were constructed using TruePrep DNA Library Prep Kit (Vazyme, TD501). 10x Genomics scRNA-seq library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, GCs 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. smart-seq3xpress scRNA-seq,10x Genomics scRNA-seq