GSM6742572: VDJ Library 3 Run 2; Mus musculus; OTHER
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 Enzyme Free Cell Dissociation Buffer (Gibco) at 37° C for 2 minutes followed by washing in Click's media containing 10% FBS. Islet cells from each animal were then resuspended in 1x PBS supplemented with 1% FBS and 0.05% sodium-azide (FACS buffer) and incubated on ice for 10 minutes with purified anti-mouse CD16/32 Fc block antibodies (BD) at a 1:200 final concentration. Total-Seq-C antibodies (Biolegend) were then added to each sample at a concentration of 1:200 and the samples were incubated on ice for 25 minutes. Finally, all of the samples were washed 2 times with 1 ml of FACS buffer and then pooled together into 1 tube for sequencing. Pooled cells were split four ways (libraries 1-4). All samples were mixed with the reverse transcription (RT) mix and partitioned into single cell Gel-Bead in Emulsion (GEM) using 10x 5' Chromium Single Cell Immune Profiling kit (10x Genomics). Single cell gene expression, cell surface protein, and T cell receptor (TCR) libraries were prepared as instructed by 10x Genomics user guides (https://www.10xgenomics.com/resources/user-guides/). All libraries were quality controlled using Bioanalyzer (Agilent, Santa Clara, CA).  Each library was sequenced twice on an Illumina NovaSeq 6000 instrument (runs 1 and 2).