Single cell RNA-seq analysis of bone marrow (BM) samples of patients with B-Cell Acute Lymphoblastic Leukemia treated with anti CD19 CAR T cells
Frozen human BM and Infusion Product (IP) cryotubes were rapidly thawed and stained with hCD45 PO (clone HI30, Invitrogen) and CD3 PerCP (clone SK7, BD) or with CD19 biotin protein (Miltenyi) followed by Biotin APC (REA746, Miltenyi) and CD3 PercP (clone SK7, BD) antibodies, respectively. BM cells were separated into CD45+CD3+ and CD45+CD3- fractions, whereas IP was separated into CD3+CAR+ and CD3+CAR- cells by flow cytometry-based sorting (BD FACS ARIA III analyser). All samples were gated based on physical parameters, followed by the exclusion of doublets and death cells (DAPI high). After sorting, cells were counted and checked for viability For each sample,cells were loaded in a 10x Genomics Chromium Next GEM Chip according to manusfacturer's protocol Libraries were constructed using 10x Genomics Chromium Next GEM Single Cell 5' v1 kit