Single-cell transcriptomic analysis of mouse lung cDC2s at various times post allergic sensitization

Conventionaldendritic cells (cDCs) in the lung that drive effector T cell differentiation, and initiate allergic responses upon inhalation of allergens. CD11b+type 2 cDCs (cDC2s) are heterogeneous, but the relationship among subpopulations has been elusive. To analyze their developmental pathways of cDC2 subpopulations, we performed single cell RNA-sequencing (scRNAS-Seq) of total lung cDC2s at various times following inhalation of house dust extracts, and detected multiple clusters based on their differential transcriptomes. The data of cDC2 transcriptome at steady state, early and later stages after activation will be applicable for the maturation pathway analysis of cDC2s. Overall design: CD11b+cDC2s (CD45+ CD11c+ I-A+ Siglec-F– CD88– F4/80– CD103– Live/Dead–) from naive C57BL/6 mice, and mice 6 and 18 hours after inhalation house dust extract together with ovalbumin werepurified by flow cytometry sorting, and labeled with barcoded antibodies then loaded into the Single Cell Chip followed by forming single cell. The Gene Expression library and the ADT Library were prepared using the Chromium single cell 3' v3 library and gel bead kit (10x Genomics) and additional reagents recommended in the protocol of Total A-seq (Biolegend). The two libraries were mixed at 10:1 molar ratio (Gene Expression library to ADT library) andsequenced by the NIEHS Epigenomics and DNA Sequencing Core Laboratory on NovaSeq 6000 (Illumina) with paired-end sequencing (Read 1: 28; Read 2: 90).