Single-cell RNA-seq of CD45-CD24- cells

Single CD45^-CD24^- cells were sorted by FACS ARIA III (BD) and collected in PBS containing 0.04% w/v BSA at a density of 400 cells/μl. Chromium™ Controller was used for partitioning single cells into nanoliter-scale Gel Bead-In-EMulsions (GEMs) and Single Cell 3’ reagent kit v2 for reverse transcription, cDNA amplification and library construction (10xGenomics, Cat. #120236). The detailed protocol was provided by 10xGenomics. SimpliAmp Thermal Cycler was used for amplification and incubation steps (Applied Biosystems). Libraries were quantified by Qubit^TM 3.0 Fluometer (ThermoFisher) and quality checked using 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent). Sequencing was performed in paired-end mode (2 × 75 cycles) on an Illumina NextSeq 500 sequencer to attain approximately 75,000 ± 25,000 reads per single cell. The scRNA-seq raw and processed data have been deposited in the NCBI GEO database under accession code GSE172526 and GSE106489 for D56 mLN-SPF^{11 (link)}.