GSM7889150: 10-weeks Cell hashing; Mus musculus; RNA-Seq
NOD mice were euthanized by CO2 asphyxiation and immediately dissected for pancreas perfusion and individual islet picking. Pancreas perfusion was performed under a dissecting microscope. The pancreatic duct was clamped using surgical clamps and 3 ml of 600 U/ml Collagenase IV (Gibco) dissolved in HBSS (Gibco) was injected using a 30G needle. Perfused pancreata were harvested and incubated at 37°C for 30 min. After the incubation, HBSS with R10 was added to quench collagenase. After washing twice with HBSS+R10, the tissue was plated on a 10 cm plate, individual islets were picked using a micropipettor. Islets were then incubated in dissociation buffer (Gibco), centrifuged, and resuspended in the staining mix (1:500 dilution of anti-Thy1.2-BV605 + 1:500 dilution of Live/Dead-APC-Cy7, and 1:100 dilution of cell hashing TotalSeq antibodies (Biolegend)). After staining, the cells were resuspended in PBS+0.04% BSA (Millipore Sigma) and sorted on BD FACS Aria III sorter. After sorting the cells, they were counted and processed for scRNAseq. Cells were processing using 10x 5' single cell gene expression kit v3 in a Chromium controller according to the manufacturer's protocols. V(D)J enrichment was done using the single cell 5' VDJ enrichment kit according to the manufacturer's protocols. Libraries were sequenced on HiSeq4000 (Novogene Inc) with a 70:20:10 mix for gene expression:VDJ:hashing libraries. scRNAseq; Individual mice are hash-tagged with TotalSeq antibodies. 3-4 mice from each time point were pooled using cell hashes, and run on a GEM. Each timepoint was processed independently through 10x protocol and through Illumina sequencing.