Determine MARCH1-dependent molecular differences in dendritic cells that carry allergen from the lungs to the draining lymph nodes

MARCH1-/- mice have a deficit in TH2 cell development in the mediastinal LN to HDM allergens. Pulmonary DCs that capture allergens and migrate to mediastinal lymph node have been implicated in driving TH2 cell polarization. We then investigated whether MARCH1 is involved in transcriptional reprograming of migratory DCs, which has been suggested to condition the DCs to drive TH2 cell development. To do this, we performed single cell RNA sequencing through the 10X Chromium platform of allergen-carrying WT or MARCH1-/- DCs in the LNs of mice. Overall design: HDM+ WT DCs or HDM+ MARCH1-/- DCs were enriched from the medLNs of CD45.1/.1 Zbtb46GFP :CD45.2/.2 Zbtb46GFP MARCH1-/- mixed BM chimeric mice (24h after oropharyngeal aspiration) and the DCs were sorted using a FACS Aria. Immediately post-sorting, cells were run on the 10X Chromium (10X Genomics) and then through library preparation by the Institute for Human Genetics at UCSF following the recommended protocol for the Chromium Single Cell 3' Reagent Kit (v3 Chemistry). Libraries were run on the NovaSeq 6000 for Illumina sequencing. Post-processing and quality control were performed by the Genomics Core Facility at the Institute for Human Genetics at UCSF using the 10X Cell Ranger package (Cell Ranger Version 3.0.2, 10X Genomics). Primary assessment with this software for WT DC sample reported 2,441 cell-barcodes with 7,651 median unique molecular identifiers (UMIs, transcripts) per cell and 2,004 median genes per cell sequenced to 61.4% sequencing saturation with 48,559 mean reads per cell. Primary assessment with this software for MARCH1-/- DC sample reported 681 cell-barcodes with 6,309 median unique transcripts per cell and 1,774 median genes per cell sequenced to 73.1% sequencing saturation with 202,410 mean reads per cell.