scRNA-seq of immune cells from pancreatic lymph nodes of NOD mice, VDJ
Single-cell suspensions from pancreatic lymph nodes and lysed spleens were labelled with the LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (Invitrogen #L34975), anti-CD45R/B220 (clone RA3-6B2, Biolegend #103248) and purified Fc receptors-blocking anti-CD16/32 (clone 2.4G2, BD Pharmingen # 553141) and one of the following hashtag antibodies: Mouse hashtag #1 (ACCCACCAGTAAGAC, Biolegend #155861), Mouse hashtag #2 (GGTCGAGAGCATTCA, Biolegend #155863), Mouse hashtag #3 (CTTGCCGCATGTCAT, Biolegend #155865), Mouse hashtag #4 (AAAGCATTCTTCACG, Biolegend #155867), Mouse hashtag #5 (CTTTGTCTTTGTGAG, Biolegend #155869), Mouse hashtag #6 (TATGCTGCCACGGTA, Biolegend #155871), Mouse hashtag #7 (GAGTCTGCCAGTATC, Biolegend #155873), Mouse hashtag #8 (TATAGAACGCCAGGC, Biolegend #155875), Mouse hashtag #9 (TGCCTATGAAACAAG, Biolegend #155877) for 30 minutes on ice.  Cells from each tissue were mixed together in one tube, washed with PBS/0.05% BSA and counted using the TC20 Automated Cell Counter (#1450102, Bio-Rad).  The cDNA libraries were prepared using the Feature Barcode technology for Cell Surface Protein protocol (#CG000186 Rev D) with the Chromium Single Cell 5 Library & Gel Bead and Chromium Single Cell 5' Feature Barcode Library kits (10x Genomics, #PN-1000014, #PN-1000020, #PN-1000080, #PN-1000009, #PN-1000084) according to the manufacturer's instructions. Sequencing was performed on NovaSeq 6000 platform (Illumina). The V(D)J library was built following the same protocol with Chromium Single Cell V(D)J Enrichment Kit, Mouse T Cell kit (10X Genomics, #PN-1000071).