title: minor salivary glands single cell RNA sequencing case 11: Sample MSG11
design: Comprehensive bioinformatics methods are described in Huang and Perez, et al., 2021. Subjects were evaluated as on NIH IRB-approved research protocols. Briefly, comprehensive oral, rheumatologic, pathological, and ophthalmologic evaluations were done. American College of Rheumatology 2016 Sjogren's Syndrome Classification criteria were applied to all subjects. Minor salivary gland biopsies were taken on all subjects and histopathological assessment for inflammation, fibrosis, and atrophy was captured. Additional glands were taken for dissociation. Tissues were sharply dissected then dissociated using the Miltenyi Multi-tissue Dissociation Kit A using the Multi_A01 in C-type tubes at 37o C in an OctoMACS tissue disruptor using heated sleeves. Crude single-cell suspensions were serially filtered through 70- and 30-um filters and rinsed with 1x Hanks' buffered salt solution. Cells were centrifuged at 300g for 10 min at 4o C and washed once with 1x Hanks' buffered salt solution. Cell counting and viability were determined using a Trypan blue exclusion assay. Single-cell suspensions targeting approximately 5,000 cells were prepared as described in Huang and Perez, et al., 2021, and loaded onto a 10x Genomics Chromium Next GEM Chip B following the manufacturer's recommendations. After cell capture, single-cell library preparation was performed following the instructions for the 10x Chromium Next GEM Single Cell 3' kit v3 (10x Genomics). The libraries were pooled and sequenced on four lanes of a NextSeq500 sequencer (Illumina), adopting the read configuration indicated by the manufacturer. Raw expression matrices were filtered, normalized and log transformed for further processing using standard SCANPY (v1.4.3) procedure. BBKNN was used for batch correction across samples, and multiple clustering rounds of different resolutions were performed to resolve subpopulations, and then manual annotations were made considering published human references. Non-SS (those who do not meet classification criteria) and SS subjects were compared for changes in gene expression per cell based microarray results to identify cell types contributing to epithelial-to-mesenchymal signature genes expression.