GSM8817946: SG TCR, DC, Female 2; Mus musculus; OTHER
Salivary glands of C57BL/6.NOD-Aec1/2 (51 weeks old, n=2 female, 2 male) and B6 (64 weeks old, n=2 female, 2 male) mice were explanted and digested in a buffer containing 1 mg/ml DNase (Sigma-Aldrich, St. Louis, MO) and 1 mg/ml Collagenase Type 4 (Worthington, Lakewood, NJ, USA) in RPMI (Lonza, Allendale, NJ) complete media (10% FBS, 2 mM L-glutamine, 0.05 mM β-mercaptoethanol). Tissues were placed in a MACS C tube (Miltenyi Biotec, San Diego, CA) for desiccation on GentleMACS V1.02 for a pulse of 38 seconds. After a 10-minute incubation at 37°C, the digest buffer was removed and placed into 4°C RPMI complete media. The process was repeated twice. Single-cell suspensions were centrifuged (2500 rpm, 10 min, 4°C) and resuspended in PBS for filtration through a 70-μm sterile cell strainer (Fisher, Pittsburgh, PA). After a wash with PBS, cells were resuspended again in PBS for lymphocyte isolation with Lympholyte-M cell separation media (Cedar Lane, Burlington, Canada) per the manufacturer's instructions. Single-cell suspensions were stained for DAPI, and live cells were sorted with a sorter (SH800S, Sony, San Jose, CA) into RPMI containing 10% FBS on ice for single-cell sequencing library preparation. A total of 8 samples were loaded onto a Chromium Next GEM Chip G, analyzed by the Chromium Controller (10x Genomics, Pleasanton, CA) for Gel Beads-in-emulsion (GEMs) generation and reverse transcription. The generated cDNA was purified with SPRIselect (Beckman Coulter Inc, Indianapolis, IN) and used for 5′ gene expression with V(D)J library construction. The cDNAs and libraries were examined for quality control using D5000 ScreenTape (Agilent Technologies, Waldbronn, Germany), and Qubit (Thermo Fisher Scientific, Waltham, MA) was used for quantification. To achieve 5000 reads per cell for the V(D)J library, the libraries were sequenced using the Illumina NovaSeq6000 system (Illumina, San Diego, CA).