alias: E-MTAB-6505:Day2_r2_p
title: NextSeq 500 sequencing; Monocyte-derived IL-1 and IL-6 are differentially required for cytokine release syndrome and neurotoxicity by CAR-T cells.
design: Monocyte-derived IL-1 and IL-6 are differentially required for cytokine release syndrome and neurotoxicity by CAR-T cells.
library_construction_protocol: In order to generate HuSGM3 CAR-T cells, newborn female or male NSG-SGM3 mice were sub lethally irradiated and injected intra-liver with 1x10^5 human cord blood-derived CD34+ hematopoietic stem cells. T cells developing in humanized NSG-SGM3 mice were retrieved from spleen and bone marrow, activated and transduced ex vivo to express CD19-targeting CAR molecule (HuSGM3 CD19 CAR-T). After transduction and expansion, HuSGM3 CD19 CAR-T cells were infused back into NSG-SGM3 mice previously co-engrafted with human cord blood-derived CD34+ hematopoietic stem cells and with ALL-CM leukaemia cells. After HuSGM3 CD19 CAR-T cell infusion, spleen from leukaemic and humanized SGM3 recipient was dissociated and cells subjects to FACS sorting. Human CD45+ cells (containing CB-derived normal hematopoiesis, ALL-CM leukaemic cells and HuSGM3 CAR-T cells) were recovered. CB CD34+ cells were supplied by commercial vendors (Lonza). HuSGM3 CAR-T cells generated as previously described. ALL-CM leukaemia cells were derived from patient with chronic myeloid leukaemia in lymphoid blast crisis.Spleen from leukemic HuSGM3 mice treated with CD19-28z chimeric antigen receptor-expressing T lymphocytes (CAR-T) cells (described here: https://www.cancer.gov/publications/dictionaries/cancer-drug/def/autologous-cd19-28z-chimeric-antigen-receptor-expressing-t-lymphocytes) was dissociated and cells subjected to FACS sorting. Human CD45+ cells were recovered. Droplet-based digital 3′ end scRNA-Seq was performed on a Chromium Single-Cell  Controller (10X Genomics, Pleasanton, CA) using the Chromium Single Cell 3′ Reagent Kit  v2 according to the manufacturer's instructions. Briefly, suspended single cells were  partitioned in Gel Beads in Emulsion (GEMs) and lysed, followed by RNA barcoding, reverse  transcription and PCR amplification (12-14 cycles). Sequencing-ready scRNA-Seq libraries were  prepared according to the manufacturer's instructions, checked and quantified on 2100  Bioanalyzer (Agilent Genomics, Santa Clara, CA) and Qubit 3.0 (Invitrogen, Carlsbad, CA)  instruments.
identifiers.namespace: San Raffaele Telethon Institute for Gene Therapy.
identifiers.id: E-MTAB-6505:Day2_r2_p