GSM8136919: NDMM4, OL; Homo sapiens; RNA-Seq
BM from healthy donors, and BM or FL samples from patients with MM, were freshly collected using either standard blind (for healthy volunteers) or CT guided biopsy (for patients). Ficoll paque was used to exclude red blood cells (RBCs). To keep neutrophils in the leukocytes, everything above the RBCs including Ficoll itself were collected. CD11b+CD3-CD56-CD138- myeloid cells were freshly isolated using a SONY flow sorter. Selected cells were immediately used for scRNA-seq. Single cell GEX libraries are generated using Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 on the 10X Genomics platform.  Cell suspensions are first assessed with ViaStain AOPI using a Cellometer K2 automated cell counter (Nexcelom), to determine concentration, viability and the absence of clumps and debris that could interfere with single cell capture.  Cells are loaded into the Chromium X Controller (10X Genomics) where they are partitioned into nanoliter-scale Gel Beads-in-emulsion with a single barcode per cell.  Reverse transcription is performed and the resulting cDNA is amplified.  The full-length amplified cDNA is used to generate gene expression libraries by enzymatic fragmentation, end-repair, a-tailing, adapter ligation, and PCR to add Illumina compatible sequencing adapters.  The resulting libraries are evaluated on D1000 screentape using a TapeStation 4200 (Agilent Technologies), and quantitated using Kapa Biosystems qPCR quantitation kit for Illumina (Roche).