Dissection by single-cell RNA-sequencing of the cellular composition of mouse salivary gland epithelia (wild-type and irradiated).

The major salivary glands (SG) are exocrine glands responsible for the physiological production of saliva, a sero-mucinous fluid that plays a key role in the digestion and swallowing of food, the articulation of speech and the maintenance of oral hygene. Among the principal causes of SG dysfunction is radiation treatment of head-and-neck malignancies, which can cause severe damage to multiple cell-types within the SG epithelium, resulting in permanent loss of saliva production and substantial disability. The aim of this study is to analyze the cellular composition of the mouse SG epithelium using single-cell RNA-sequencing (scRNAseq) in order to: 1) gain a deeper understanding of the rich repertoire of different epithelial cell-types that coexist witin it; and 2) to elucidate whether different sub-types of epithelial cells display different radiosensitivity and are preferentially elimininated from SGs following radiotherapy. The dataset includes data from scRNAseq experiments performed on the epithelial compartment (Epcam+) of mouse adult sub-mandibular glands (C57BL/6J, female), either at baseline (wild type) or following stereotactic X-ray irradiation (30 Gy). In the specific case of wild-type mice, the dataset also includes data from a secind scRNAseq experiment performed on the basal compartment (Epcam+, Cd49f-high) of the sub-mandibular gland, which is postulated to contain a minority population of epithelial cells with stem-progenitor properties. Overall design: The cell populations included in the study were sorted by fluorescence activated cell sorting (FACS), starting from the SMG of adult (8-10 weeks) female C57BL/6J mice (The Jackson Laboratory: stock #000664). Each scRNAseq experiment was representative of 3 independent biological replicates (3 independent mice) that were sorted in parallel and labelled with oligonucleotide-tagged antibodies for CITE-seq applications, before being pooled. Mouse SMG were surgically dissected, mechanically disaggregated by mincing into small fragments and then dissociated into single-cell suspensions using DNAse-I (100 units/ml), Collagenase-III (200 units/ml), Hyaluronidase (100 units/ml) for 2 hours, followed by a brief digestion (5 minutes) with Trypsin (0.25%). Single-cell suspensions were stained with monoclonal antibodies against seven differentially expressed surface antigens (Epcam, Cd49f, Kit, Cd3, Cd16/Cd32, Cd31, Cd45) and sorted using a FACSAria-III machine (Becton Dickinson), after exclusion of dead cells (DAPI+), cell doublets (serial gating based on FSC-A vs. FSC-H and SSC-A vs. SSC-H profiles) and cells of non-epithelial lineages (Cd3+, Cd16/Cd32+, Cd31+, Cd45+). In the specific case of the scRNAseq experiment performed on irradiated SGs, the specimens were collected 90 days after selective stereotactic irradiation (30 Gy) of the left submandibular gland, performed under Computed Tomography (CT) guidance (2 Gy/minute) using a Small Animal Radiation Research Platform (SARRP; Xstrahl). HTO barcode sequence hashtagged samples TotalSeq™ - B0301 anti-mouse Hashtag 1 Antibody (BioLegend) ACCCACCAGTAAGAC replicate 1 TotalSeq™ - B0302 anti-mouse Hashtag 2 Antibody (BioLegend) GGTCGAGAGCATTCA replicate 2 TotalSeq™ - B0303 anti-mouse Hashtag 3 Antibody (BioLegend) CTTGCCGCATGTCAT replicate 3