SmartSeq2 manual scRNA-sequencing of salivary gland tissues isolated from Sjögren's cases and individuals with non-Sjögren's sicca syndrome.
Cells were processed using an adapted Smart-Seq2 method, as described by Eltahla et al. (67). Briefly, minor salivary gland CD3+CD4+CD45RA– cells were bulk-sorted using a FACSAria™ Illu (BD Biosciences), then singly sorted into 96-well PCR plates containing 4 μL/well of catch buffer (RNAsin, 0.2% Triton-X 100, 10mM dNTPs, 5 mM oligo-dT30-VN) using a MoFlo XDP (Beckman Coulter). Plates were immediately sealed and stored at -80°C. Cells were then processed by plate to yield full-length cDNA, which was used to prepare sequencing libraries using the Nextera XT (Illumina) workflow. Single cell libraries were pooled, then sequenced with paired end 150 bp reads to a target depth of 0.5 x 106 reads/cell on an Illumina Novaseq 6000.