Illumina NovaSeq 6000 sequencing; scRNAseq of the bone marrow immune microenvironment in control and 5TGM1 myeloma-bearing C57Bl/6 and KaLwRij mice
scRNAseq of the bone marrow immune microenvironment in control and 5TGM1 myeloma-bearing C57Bl/6 and KaLwRij mice
1 femur and 2 tibias from each mouse were used. After isolating and cleaning the bones, one end was cut off and bones were placed into a 0.5 ml microcentrifuge tube with a hole punched in the bottom. This was nested into a 1.5 ml microcentrifuge tube and spun down at 10,000 RPM for 15 seconds to collect the bone marrow. Following resuspension in PBS containing 1% FCS and 1 mM EDTA, erythrocytes were lysed. Remaining cells were washed with PBS containing 2% FCS, filtered through a 100 um cell strainer and counted. Following erythrocyte lysis, the samples were filtered through a 100 um cell strainer and counted. Fc-receptors were blocked with 10% normal mouse serum and anti-CD16/32 (BD, clone 2.4G2) prior to incubation with the following antibodies: CD3e-PerCpCy5.5 (eBioscience, clone 145-2C11), CD8-FITC (BD, clone 53-6.7), CD11b-APCeFluor780 (eBioscience, clone M1/70), CD19-AF700 (eBioscience, clone eBio1D3), CD27-BV510 (Biolegend, clone LG.3A10), CD44-BV510 (Biolegend, clone IM7), NKp46-PE-Cy7 (eBioscience, clone 29A1.4), Gr1-APC (BD, clone RB6-8C5), Ly6c-BV510 (Biolegend, clone HK1.4). Samples were incubated for 25 minutes on ice in the dark. Following staining, cells were washed and resuspended in PBS + 2% FCS containing DAPI, and sorted with a BD FACS Aria III. Sorted T, NK, B, monocyte/macrophages and neutrophil granulocytes were combined per mouse and processed as one sample. Single cells were encapsulated for cDNA synthesis and barcoded using the Chromium Single Cell 3' Reagent Kit v3 (10X Genomics) Libraries were constructed according to the manufacturerâ€™s recommendations (10X Genomics)