GSM6997591: Pancreatic islets T cells, TCR-derived cDNA, biol rep1; Mus musculus; RNA-Seq
Islets were harvested from NOD female mice and labelled with anti-TCRvb (BioLegend, APC, clone H57-597, 1:300), anti-CD4 (BioLegend, TotalSeq-C0001, clone RM4-5, barcode- AACAAGACCCTTGAG, 1:1000), anti-CD8b-biotin (BioLegend, clone YTS156.7.7, 1:8000), anti-CD62L (BioLegend, TotalSeq-C0112, clone MEL-14, barcode- TGGGCCTAAGTCATC, 1:1000), and anti-CD11a (BioLegend, TotalSeq-C0595, clone M17/4, barcode AGAGTCTCCCTTTAG, 1:1000) primary antibodies followed by secondary labelling with PE SA (BioLegend, TotalSeq-C0951, barcode- AACCTTTGCCACTGC, 1:8000). DAPI was used to exclude dead cells while sorting. Live TCR+ cells were sorted from the islets on an Aria sorter and resuspended at 1000 cells/ul in PBS with 0.04% bovine serum albumin Libraries were generated using Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) with Feature Barcode technology for Cell Surface Protein and Immune Receptor Mapping. All amplification steps used a ThermoFisher Proflex System thermocycler. The resulting GEM's were transferred to new tubes for GEM-RT. After the GEM-RT incubation the samples were cleaned-up and readied for cDNA amplification using 11 cycles of amplification. Amplified cDNA was size-selected using SPRIselect Beads (BECKMAN COULTER). The quality and concentration of the isolated cDNA was determined using a Fragment Analyzer (Agilent). The resulting cDNA was used to generated the various library types; V(D)J TCR,  5 ' Gene Expression (GEX) and Cell Surface Receptor.