title: Illumina NovaSeq 6000 paired end sequencing; Single-cell transcriptomic analysis of NK cell dynamics in myeloma patients reveal persistent reduction of cytotoxic NK cells from diagnosis to relapse
design: Single-cell transcriptomic analysis of NK cell dynamics in myeloma patients reveal persistent reduction of cytotoxic NK cells from diagnosis to relapse
library_name: MM23_p
library_construction_protocol: BM aspirates from patients with newly diagnosed MM were collected at the Erasmus Medical Centre (Rotterdam, the Netherlands) as part of inclusion into Dutch-Belgian Cooperative Trial Group for Hematology Oncology (HOVON) trials or European Myeloma Network (EMN) trials. The protocols were approved by the Institutional Review Board of the Erasmus Medical Centre (Rotterdam, The Netherlands). Patient samples were obtained after informed consent, in accordance with the Declaration of Helsinki. Control BM was obtained by sternal aspiration from donors undergoing cardiothoracic surgery or by manual BM collection from femur heads collected after hip replacement surgery. Bone marrow mononuclear cells (BMMCs) were isolated by density gradient centrifugation using Histopaque (Sigma-Aldrich) and subsequent plasma cell isolation was performed using magnetic CD138+ bead enrichment (Stemcell). Plasma cells, pre-isolation fractions and plasma-cell depleted fractions were viably cryopreserved in 10% dimethyl sulfoxide (DMSO, Sigma-Aldrich). The 10X Chromium Single cell 3' reagent Kit v3 (10X Genomics) was used for construction of single-cell transcriptomic libraries. Reverse transcription, cDNA amplification, and library preparation were all performed according to the manufacturer's instructions. Cryopreserved bone marrow aspirates from 6 NDMM, 21 post-therapy and 6 RRMM patients were thawed and sorted into CD45+CD38−CD34−CD235a−CD71− and CD45+CD38+CD34−CD235a−CD71− immune fractions and processed for single-cell RNA sequencing according to the 10X Genomics protocol as previously described. This dataset was supplemented with data from 5 controls and 13 additional NDMM patients that were previously generated in our lab (De Jong et al. Nature Immunology 2021), resulting in a total single-cell dataset of 51 individuals, including 19 NDMM patients and 6 RRMM patients. As the majority of NK cells are CD38+and CD38high NK cells have increased cytotoxic function against cancer cells, CD38+ NK cells were identified in silico by transcription of KLRF1, KLRD1, GNLY and NKG7 as previously described (Yang et al. Nature Communications 2019).