GSM6836812: 6434_B2IKC; Mus musculus; OTHER
scRNA-seq + scTCR-seq were performed in 13 bone marrow aspirates from 2 healthy (Ycɣ1), 3 MGUS (BIcɣ1), 3 MM (BIcɣ1), 3 MGUS (MIC) and 2 MM (MIC) bearing mice. Cells were FACS sorted (a mix of 0.8x10^5 T cells + 0.8x10^5 NK cells + 0.25x10^5 monocytes + 0.15x10^5 B cells) in 100 µL of PBS+0.05% BSA. Samples with at least 90% viability were processed using the 10X Genomics (CA, USA) scRNA/TCRseq kit, following the manufacturer's instructions (Chromium Next GEM Single Cell V(D)J v1.1 protocol rev F for human samples and Chromium Next GEM Single Cell 5' v2 Dual Index protocol rev B for mice samples). Quality control was performed with Qubit Fluorometric Quantification (ThermoFisher Scientific, MA, USA) using the double-stranded DNA high-sensitivity assay kit, and with TapeStation (Agilent, Santa Clara, CA) using high-sensitivity screentapes. Libraries were sequenced on a NextSeq 550 (Illumina, San Diego, CA). WES was performed in 71 BM samples isolated from GFP+ CD138+B220− PCs (purity, >99%), including 62 samples from the MM stage, 3 samples of pooled PCs from nine mice at the MGUS stage (3 mice with similar genotype were included on each pooled sample), and 6 samples from MM-derived cell lines. As MM reference controls, 5TGM1 and 12598Vk*Myc cell lines were also characterized. Library was performed according to the manufacter's instructions (Chromium Next GEM Single Cell 5' v2 Dual Index protocol rev B, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. Of note, according to TCR-seq protocol from manufacturer, we further sequenced (after specific amplification) CDR3 sequences from each CD3+ T cells. scRNA-seq, scTCR-seq, WES