Single cell mRNA sequencing of Peyer's patch LysoDCs and cDC2s and blood CD45+CD115hi cells

We investigated the genetic relatedness between CD115hi blood cell subsets, LysoDCs and Peyer's patch cDC2s, a close but ontogenically unrelated phagocyte subset. We isolated Ly6ChiCD43-, Ly6CloCD43hi and Ly6C-CD43-CD11b+SIRPa+ cells from CD115hi blood cells of 9-day clodronate-treated mice, when the latter were in higher number than non-treated mice. In parallel, we sorted cDC2s and the four differentiation states of LysoDCs from PPs of non-treated mice. scRNAseq analysis of these populations allowed to identify the blood precursors of LysoDCs. Overall design: In the first dataset CD115hi cells were sorted from the blood of 20 lipoclodronate-treated C57BL/6Rj female mice and subsets of cDC2s and LysoDCs sorted from PPs of 30 untreated C57BL/6Rj female mice. 4,400 follicular LysoDCs, 3,800 immature LysoDCs, 6,000 intermediate LysoDCs, 2400 mature LysoDCs, 6,000 immature cDC2s, 1,500 maturing cDC2s, 6,000 Ly6Chi monocytes, 6,000 CD43hi monocytes and 15,000 DN CD115hiLy6C-CD43-CD11b+SIRPa+ blood cells were tagged 10 minutes with Hashtag Oligonucleotides (HTOs) from Biolegend (TotalSeq™-B) and resuspended in PBS+0.04%BSA. In the second dataset LysoDCs subsets were extracted from 20 untreated C57BL/6Rj female mice and blood monocyte subsets from 18 lipoclodronate-treated C57BL/6Rj female mice. Cells were tagged with HTOs before the sort to decrease the cell mortality observed in the first dataset. CD11c-enriched phagocytes were split in 4 and tagged with HTOs. Blood cells were split in 3 according to the blood ratio of Ly6Chi monocytes, CD43hi monocytes and candidate precursors (either CCR2- or CCR2+) and tagged with HTOs. After the sort, cells were resuspended with PBS+0.04%BSA.