Single cell analysis of DC patient
Single-cell capture and downstream library construction were performed using Chromium Next GEM Single Cell 3 Reagent Kits v 3.1 (10x Genomics; PN-1000121, PN-1000127 and PN-1000213) for PBMCs from the DC patient and age-matched controls, according to the manufacturers protocol. Briefly, a total of approximately 8600 single cells, 50l of barcoded gel beads and 45l partitioning oil were loaded into Chromium Next GEM Chip G to generate nanoliter-scale gel beads-in-emulsion (GEMs). Afterwards, the polyadenylated mRNAs were reverse transcribed inside each GEM, and the full-length cell-barcoded cDNA was amplified via PCR to generate sequencing libraries. Library quality was assessed by using the Qubit 4.0 Fluorometer (Thermo Scientific) and a Qsep100TM system (Bioptic, Taiwan) to determine the library concentration and library size, respectively. In general, fragments of approximately 450-500 bp in size were expected for single-cell 3 gene expression libraries. The effective concentrations of the library were assessed by Q-PCR. The qualified libraries were pooled according to the effective concentration and expected data volume. The library was sequenced on Illumina NovaSeq 6000 sequencers according to read length: 28 bp Read 1 (16 bp single-cell barcode, 10x barcode; 12 bp unique molecular identifier, UMI), 91 bp Read 2 (transcript insert or feature barcode in the case of the cell hashing library), and 8 bp i7 Index (sample index). TruSeq Read 1 and TruSeq Read 2 are standard Illumina sequencing primer sites used in the paired-end sequencing of single-cell 3 gene expression libraries.