scRNA-seq mouse bone marrow KO stromal normal control
Single cells were barcoded using the 10 Chromium single-cell platform, and cDNA libraries were prepared following the manufacturers instructions (Chromium Single Cell 3 Kits v3.1, 10 Genomics, USA). Bone marrow cells were harvested from 5-10 WT or Lxn-/- mice (either normal or AML mice). Stromal cells were sorted by cell surface marker CD45-, Lin-, Ter119-, and 7-AAD. Live cell counts were further determined using a hemocytometer. Libraries were sequenced on the NovaSeq 6000 (Illumina) platform (paired-end 150bp). The sequencing reads were aligned to the mouse reference genome (mm10) and quantified using CellRanger (v5.0.0, https://www.10xgenomics.com) with default parameters. The filtered count matrix, features, and barcodes of each sample from CellRanger were imported into Seurat (v4.3.0) to create Seurat objects. To accurately characterize the transcriptomic changes of bone marrow and stromal cells at single-cell level in Lxn-/- mice, the cells that meet the following criteria were removed: (1) with less than 1000 or more than 7500 detected features; (2) with mitochondrial transcripts that accounted for more than 10% of the total detected transcripts; (3) predicted to be doublets by Scrublet (v0.2.3). Moreover, the genes detected in less than three cells for each sample were also excluded in the following analysis. All 8 samples (HSPCs and stromal cells before transplantation, GFP+ Mll-Af9 cells and stromal cells after transplantation) were merged together to perform principal component analysis (PCA) and dimensionality reduction analysis. Different HSPC and stromal populations were annotated with known lineage-specific and stromal markers. Cell-cell communication differential analysis was performed with CellChat.