ScRNA-seq of CD45 positive cells

Bone marrow derived cell were isolated from C57BL/6 mice tibia and femur using an aseptic technique. The bone marrow was flushed out of the bone marrow cavity by using a 26-gauge needle with 10ml PBS. Subsequently cell suspension was passed through a 70µm nylon mesh. After a 10 min centrifugation step at 500 x g, cells were incubated in 2ml red cell lysis buffer for red blood cell removal. After 4 min incubation time, lysis was stopped by adding 23 ml PBS. Cells were resuspended in PBS and FACS sorted for CD45 cells using an AriaIII-sorter and 7-AAD as indicator for dead cells. The cell suspensions were counted with Moxi cell counter and diluted according to manufacturer’s protocol to obtain 10.000 single cell data points per sample. Each sample was run separately on a lane in Chromium controller with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10xGenomics). Single cell RNAseq library preparation was done using standard protocol. Sequencing was done on Nextseq500 and raw reads were aligned against the mouse genome (mm10) and counted by StarSolo (44) followed by secondary analysis in Annotated Data Format. Preprocessed counts were further analyzed using Scanpy (45). Basic cell quality control was conducted by taking the number of detected genes and mitochondrial content into consideration. We removed 214 cells in total that did not express more than 200 genes or had a mitochondrial content less than 3%. Further, we filtered genes if they were detected in less than 30 cells (<0.3%). Raw counts per cell were normalized to the median count over all cells and transformed into log space to stabilize variance. We initially reduced dimensionality of the dataset using PCA, retaining 50 principal components. Subsequent steps, like low-dimensional UMAP embedding (McInnes & Healy, https://arxiv.org/abs/1802.03426) and cell clustering via community detection (Traag et al., https://arxiv.org/abs/1810.08473), were based on the initial PCA. Final data visualization was done by cellxgene package.