NextSeq 500 sequencing; scRNA-seq of mouse hematopoietic stem cells after 4 hours of LPS/PBS treatment in vivo.
scRNA-seq of mouse hematopoietic stem cells after 4 hours of LPS/PBS treatment in vivo.
Mice were treated intraperitoneally with 35 ug of LPS or PBS and sacrificed 4 hours after the treatment. Tibia, femur and hip bones were isolated from both hind legs and cleaned from tissue. Cells were isolated by crunching the BM with pestle and mortar and filtered through nylon mesh. The cell suspension was treated with ACK to remove erythrocytes. Next, the BM cells were stained with mix of lineage antibodies (CD3e, B220, Ter119, Gr-1 and CD11b) and separated on autoMACS separator to enrich for immature populations. The sorted unstained fraction was further stained with c-Kit, Sca-1, CD48 and CD150 antibodies and sorted on BDinflux machine. In total, 16000 cells were pooled for each experimental condition. WT C57BL6 mice were treated intraperitoneally with 35 ug of LPS from Escherichia coli 0111:B4 (InvivoGen, San Diego, CA, USA) or PBS control. Software cellranger (10X Genomics) version 6.1.1 was used to demultiplex fastq files from Illumina bcl files with default parameters. Resulting per-lane fastq files were merged into single file per sample. First-strand cDNA was extracted from cell suspension during library preparation routine using Chromium next gem single-cell 3′ reagent kit (version 3.1). Single-cell RNA-seq libraries were prepared using Chromium controller instrument and Chromium Next Gem single-cell 3' reagent kit version 3.1 (both 10X Genomics, Pleasanton, CA, USA) according to the manufacturer's protocol targeting 4000 cells per sample.