Single-cell RNAseq analysis (10X Genomics Chromium) of refilled lung interstium macrophages at the time points of 12h, 24h and 48h after depletion

Lung interstitium macrophages (IMs) are non-alveolar resident tissue macrophages which contribute to the lung homeostasis. These cells were reported to be heterogeneous by our group and other teams, which contains two main distinct subpopulations: CD206+ IMs and CD206- IMs. However, the exact origin of IMs and the transcriptional programs that regulate IM differentiation remains unclear. In recent report, we analyzed the refilled IMs in the course of time after induced IM depletion with single-cell RNA sequencing (10X Genomics Chromium) and bulk RNA sequencing. The lung IMs and monocytes from the mice at 12 hours (DT12h), 24 hours (DT24h) and 48 hours (DT48h) after diphtheria toxin (DT)-induced IM depletion were analyzed and compared using single-cell RNA sequencing. A subpopulation was found to be a transit differentiating cells from monocytes to IMs. Transcription factor activity analysis and trajectory showed cMAF and MAFb transcription factors played important roles in monocyte-IM differentiation. Overall design: The lung IMs and monocytes were sorted from three groups of mice: Group “DT12h” contains 5 IM-DTR mice which were treated with 50 ng diphtheria toxin (DT) 12 hours before sacrifice. Group “DT24h” contains 5 IM-DTR mice which were treated with 50 ng DT 24 hours before sacrifice. Group “DT48h” contains 5 IM-DTR mice which were treated with 50 ng DT 48 hours before sacrifice. Cells from each group were barcoded with different Biolegend anti-mouse Hastags before being pooled for library construction. Processed data then were analyzed with Bioconductor and Seurat package. The cells from different time points were demultiplexed with the barcode detected in each cell.