Unknown,Transcriptomics,Genomics,Proteomics

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Single-cell RNA sequencing of mouse primary B cells to investigate subpopulation markers

ABSTRACT: The project aims at linking functionality to metabolic fluxes of mouse primary B cells, potentially correlating antibody secretion rates with metabolic differences in subpopulations of B cells. In previous experiments, subpopulations of primary B cells with different phenotypes were observed. Therefore, the next step is to further characterize these subpopulations of B cells via single-cell RNA sequencing so that we gain insight about cell characteristics and individual gene markers, and their potential biological role. Our hypothesis is that these cells present different expression profiles of key genes and/or are relevant in different immune-related pathways.

INSTRUMENT(S): NextSeq 2000

ORGANISM(S): Mus musculus

SUBMITTER: Daniela Rodrigues

PROVIDER: E-MTAB-13205 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Memory B cells play a fundamental role in host defences against viruses. This dataset aimed at understanding their maturation and stability in the context of SARS-CoV-2 infection and consist of a longitudinal single-cell and repertoire profiling of the B cell response up to six months in four severe COVID-19 patients. All four patients were recruited at Henri Mondor University Hospital (AP-HP, Paris France), between March and May 2020, and required oxygen as treatment. Clinical and biological characteristics of these patients are summarized in the Patient_information.csv file. Peripheral (CD3-CD14-CD15-CD56-CD19+IgD-) B cells were FACS-sorted (MA900, Sony) in PBS/0.08% FCS from 4 patients (S-CoV) at baseline (M0) and 6 months (M6). 5x104 to 10x105 cells were obtained for each subset and 20000 were loaded in the 10x Chromium Controller to generate single-cell gel-beads in emulsion. The scRNA-seq libraries were generated using the Chromium Next GEM Single Cell V(D)J Reagent Kit v.1.1 with Feature Barcoding (10x Genomics) according to the manufacturer’s protocol. PBMCs were initially isolated from venous blood samples via standard density gradient centrifugation and used after cryopreservation at -150°C. Cells were thawed using RPMI-1640 (Gibco) 10% FBS, washed twice and incubated with 10µg of the SARS-CoV-2 his tagged spike protein in 100µL of PBS (Gibco) 2% FBS during 20 minutes on ice. Cells were washed and resuspended in the same conditions, then fluorochrome-conjugated antibody cocktail including the 2 anti-His was added at pre-titrated concentrations for 20 min at 4°C and viable cells were identified using a LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific) incubated with conjugated antibodies used for cell sorting (CD3/CD14/CD15/CD56/CD19/IgD) as well as a panel of barcoded TotalSeqC® and homemade anti-His antibodies (see feature_reference.csv.gz files). Three distinct sorts were performed for each donor: two at the M0 time-point (M0_Sort1 and M0_Sort2) and one at the M6 time-point (M6_Sort1). His-tagged-Spike + barcoded-anti-his staining was only included in the last two sorts (M0_Sort2 and M6_Sort1). For these two sorts it should additionally be noted that 5’-transcriptomic and ADT libraries were sequenced in two separate runs (Run1 and Run2) to achieve sufficient sequencing depth for both libraries. Both runs were pooled at the Cell Ranger analysis step.

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Single-cell RNA sequencing of mouse primary B cells to investigate subpopulation markers

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