The lymph node was removed from the collected fourth gland. The gland was mechanically minced with a scalpel and incubated with agitation in the digestion buffer [1.5 mg/mL DNAse I (#10104159001, Millipore Sigma, Burlington, MA), 0.4 mg/mL Collagenase IV (CLS-4, Lot: 47E17528A, Worthington Biochemical Corporation, Lakewood, NJ), 5% fetal bovine serum, 10 mM HEPES in Hank’s buffered salt solution] at 37 °C for about an hour until dissociated. Then, samples were pipetted and strained through a 70 μm cell strainer. Ammonium-chloride-potassium lysis buffer was used to remove residual red blood cells and dead cells were removed using Dead Cells Removal Microbeads (Miltenyl Biotec, Bergisch Gladbach, Germany) to ensure the sample viability (> 80%) for scRNAseq.
Cells were then loaded onto the Chromium Controller (10× Genomics, Pleasanton, CA), targeting 2000–5000 cells per lane. The Chromium v2 single-cell 3′-RNAseq reagent kit (10× Genomics) was used to partition cells into gel bead-in emulsions and subsequently generate the sequencing libraries according to the manufacturer’s protocol73 (link). The libraries were sequenced with a Hiseq 2500 (Illumina, San Diego, CA) with a depth of 50k–100k reads per cell. Raw sequencing data were processed using the 10× Genomics Cell Ranger pipeline (version 2.0) and aligned to the mm10 mouse genome.
Cells were then loaded onto the Chromium Controller (10× Genomics, Pleasanton, CA), targeting 2000–5000 cells per lane. The Chromium v2 single-cell 3′-RNAseq reagent kit (10× Genomics) was used to partition cells into gel bead-in emulsions and subsequently generate the sequencing libraries according to the manufacturer’s protocol73 (link). The libraries were sequenced with a Hiseq 2500 (Illumina, San Diego, CA) with a depth of 50k–100k reads per cell. Raw sequencing data were processed using the 10× Genomics Cell Ranger pipeline (version 2.0) and aligned to the mm10 mouse genome.