GSM7459093: Patient 10  post CAR  PB SC; Homo sapiens; RNA-Seq
For single cell sequencing, mononuclear cells from bone marrow (BMMCs) and peripheral blood (PBMCs) were isolated following standardized operating procedures using Leucosep tubes (Greiner Bio One, Frickenhausen, Germany) with a Ficoll gradient (Ficoll-Paque™ PLUS, VWR Avantor, Spring House, Pennsylvania). Cells were frozen using a CoolCell LX container (Corning, USA) and stored in the vapour phase of liquid nitrogen in an Askion HS200S freezer (Askion, Gera, Germany). Prior to single cell staining and library construction, samples with initial viability < 80% were subjected to dead cell removal using Viahance™ from BioPAL, Inc. following the manufacturer's instructions. A custom panel of61oligonucleotide-coupled antibodies directed against cell surface proteins was used for surface proteome analyses. The oligonucleotides consist of a linker to the antibody, a polymerase chain reaction (PCR) handle, an antibody-specific barcode and a binding site for the cell identifying capture oligonucleotide. The antibody-oligonucleotide conjugate was afterwards incubated with the cells followed by the removal of unbound antibody. After single cell droplet encapsulation, the antibody-bound oligonucleotides act as synthetic transcripts and were then processed with 10x Genomics 5' v2 chemistry to generate libraries for BCR, TCR, gene expression and surface protein expression. scRNA + surface protein expression seq, BCRseq, TCRseq