title: GSM6720608: FUN-2r; islets from 20% HC fed NOD.SCID mouse; Mus musculus; RNA-Seq
library_construction_protocol: Mice were killed by cervical dislocation and the pancreata were perfused with 5 mL of collagenase P solution [0.5 mg/ml, (Roche) in HBSS (Gibco) supplemented with 0.1 M HEPES, 1 mM MgCl2, 5 mM D- glucose, 1.25 mM CaCl2, and 0.02% BSA] via the common bile duct. Perfused pancreata were then dissected away from the surrounding tissue and incubated for 10 minutes at 37°C. Digestion was halted by washing the tissue twice with supplemented HBSS. The tissue was then applied to a Histopaque-1119 (Sigma) gradient, and the interface was collected, washed in supplemented HBSS, and then placed in Click's medium with 5% FBS. Islets were handpicked using an inverted microscope. Handpicked islets were dissociated by incubation in Trypsin-EDTA (Gibco) at 37° C for 2 minutes followed by washing in Click's media containing 10% FBS. Single cell suspensions from each mouse were sequenced individually. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, islet single cell suspensions 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.