Chromium platform

Single-cell suspensions were isolated from mouse SMG as previously reported [7 (link)]. Briefly, SMGs were minced and digested for 1 h with RPMI 1640 medium containing 1 mg/mL collagenase IV, 5 mM CaCl₂, 50 mg DNase I, and 8% fetal bovine serum with continuous shaking at room temperature. scRNA-seq libraries were generated in TAMU Genomics Core using the 10× Chromium platform and the version 3 Chromium Single Cell 3′ reagent kit following the manufacturer’s instructions (10× Genomics, Pleasanton, CA, USA). Samples were pooled in equimolar concentrations and sequenced on a single lane of an S4 2 × 150 PE flow cell on an Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA). Bioinformatics analyses were performed by researchers blinded to the treatments using the methods detailed in our recent paper [7 (link)]. The uniform approximation and projection method (UMAP) was used to visualize the clusters. The normalized data are shown as feature plots. Approximately 18,000 cells were sequenced at a depth of 1420 median genes per cell. The heatmap of S1P-related genes in major types of SMG cells was generated with globally distinguishing fold-change data exported from the Loupe Browser (Version 6.2.0, 10× Genomics, Pleasanton, CA, USA).

For single-nucleus sequencing, primary somatosensory cortical tissue from six-month Ts65Dn and CTL (n = 3 per condition) mice were microdissected and snap frozen in liquid nitrogen. Frozen tissue was thawed in 1 ml buffer HB (0.25 m sucrose, 25 mm KCl, 5 mm MgCl₂, 20 mm Tricine-KOH pH 7.8, 0.15 mm spermine tetrahydrochloride, 0.5 mm spermidine trihydrochloride, 1 mm DTT). The tissue was transferred to a 7-ml dounce; 340 μl 5% IGEPAL CA-630 (Sigma-Aldrich) and 4 ml HB were added to the tissue and the tissue was homogenized with a tight pestle 10–15 times. The sample was transferred to a 15-ml tube and total solution brought to 10 ml with 50% iodixanol; 1 ml 30% iodixanol was layered on top of 1 ml 40% iodixanol in a separate Corex tube (ThermoFisher). The 9 ml sample was layered on top of the iodixanol cushion. The sample was spun at 10,000 × g for 18 min; 1 ml of sample at the 30–40% iodixanol interface was collected. After counting nuclei with a hemocytometer, the sample was diluted to 100,000 nuclei/ml with 30% iodixanol (with RNasin) and subjected to single nuclear droplet encapsulation with the 10× Chromium platform (10× Genomics). Libraries were sequenced using the Illumina NovaSeq 6000 S4 platform at The Center for Applied Genomics (TCAG) at The Hospital for Sick Children in Toronto (ON, Canada).

MACS-enriched brain myeloid cells were subjected to single-cell library preparation. For each sample approximately 12,000 cells were washed and resuspended in PBS containing 3% FBS and immediately processed as follows. Single-cell capture, barcoding and library preparation were performed using the 10X Chromium platform (10X Genomics), using version 3 chemistry according to the manufacturer’s protocol (10X Genomics #CG00052). The resulting cDNA and indexed libraries were checked for quality on an Agilent 4200 TapeStation, quantified by KAPA qPCR, and pooled for sequencing on 16.67% of lane of an Illumina NovaSeq 6000 S2 flow cell, targeting 6,000 barcoded cells with an average sequencing depth of 50,000 reads per cell. Illumina base call (bcl) files for the samples were converted to FASTQ files using CellRanger bcl2fastq (version 2.20.0.422, Illumina).