Illumina NovaSeq 6000 sequencing; Paired scRNAseq of the and non-hematopoietic and the immune microenvironment of aspirates from newly diagnosed Multiple Myeloma patients versus non-cancer controls
Paired scRNAseq of the and non-hematopoietic and the immune microenvironment of aspirates from newly diagnosed Multiple Myeloma patients versus non-cancer controls
Viably frozen bone marrow aspirates were thawed in DMEM + 10% FCS according to the 10X Genomics protocol 'Fresh Frozen Human Peripheral Blood Mononuclear Cells for Single Cell RNA sequencing'. Only samples containing >200x10E6 MNCs were included. Fc receptors were blocked in 10% normal human AB serum (Sigma-Aldrich). To enrich for stromal fibroblasts, samples were incubated with biotinylated antibodies against CD45 (clone HI30, BioLegend), CD235a (clone HIR2, BioLegend) and CD38 (from human CD38 MicroBead Kit, Miltenyi Biotec) followed by depletion using magnetic anti-biotin beads (Miltenyi Biotec) and the iMag (BD Biosciences). After depletion, cells were stained for sorting in PBS containing 2% FCS at 4°C with the following antibodies: CD271-PE (ME20.4), CD235a-PECy7 (HI264), CD31-APCCy7 (WM59), CD44-BV711 (IM7), streptavidin-BV421 (all BioLegend), CD34-PECF610 (4H11, eBioscience), CD45-APC (2D1, eBioscience), CD38-FITC (MHCD3801, Life Technologies), CD71-Alexa Fluor 700 (MEM-75, ExBio) and CD105-BV510 (266, BD Biosciences). 7AAD (Beckman Coulter) or DAPI (Life Technologies) were used for dead cell exclusion. Cells were sorted in DMEM + 10% FCS using a FACSAria III (BD Biosciences). Prior to depletion, 10x10^6 cells were used to sort CD45+CD38- and CD38+ hematopoietic populations using the same antibody panel. Sorted CD45- cells were combined with sorted CD45+CD38- cells 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 manufacturer's recommendations (10X Genomics)
Experimental Factor: disease
multiple myeloma
Experimental Factor: immunophenotype
CD45+CD38- hematopoietic cells