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FB5P-seq: FACS-based 5-prime end single-cell RNAseq for integrative analysis of transcriptome and antigen receptor repertoire in B and T cells

ABSTRACT: FB5P-seq: FACS-based 5-prime end single-cell RNAseq for integrative analysis of transcriptome and antigen receptor repertoire in B and T cells

PROVIDER: PRJNA564993 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10099487_1.fastq.gz	Fastqsanger.gz
	SRR10099487_2.fastq.gz	Fastqsanger.gz
	SRR10099488_1.fastq.gz	Fastqsanger.gz
	SRR10099488_2.fastq.gz	Fastqsanger.gz
	SRR10099489_1.fastq.gz	Fastqsanger.gz

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FB5P-seq: FACS-based 5-prime end single-cell RNAseq for integrative analysis of transcriptome and antigen receptor repertoire in B and T cells

Project description:Single-cell RNA sequencing (scRNAseq) allows the identification, characterization, and quantification of cell types in a tissue. When focused on B and T cells of the adaptive immune system, scRNAseq carries the potential to track the clonal lineage of each analyzed cell through the unique rearranged sequence of its antigen receptor (BCR or TCR, respectively), and link it to the functional state inferred from transcriptome analysis. Here we introduce FB5Pseq, a FACS-based 5’-end scRNAseq method for cost-effective integrative analysis of transcriptome and paired BCR or TCR repertoire in phenotypically defined B and T cell subsets. We describe in details the experimental workflow and provide a robust bioinformatics pipeline for computing gene count matrices and reconstructing repertoire sequences from FB5Pseq data. We further present two applications of FB5Pseq for the analysis of human tonsil B cell subsets and peripheral blood antigen-specific CD4 T cells. We believe our novel integrative scRNAseq method will be a valuable option to study rare adaptive immune cell subsets in immunology research.

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FB5P-seq: FACS-Based 5-Prime End Single-Cell RNA-seq for Integrative Analysis of Transcriptome and Antigen Receptor Repertoire in B and T Cells.

Project description:Single-cell RNA sequencing (scRNA-seq) allows the identification, characterization, and quantification of cell types in a tissue. When focused on B and T cells of the adaptive immune system, scRNA-seq carries the potential to track the clonal lineage of each analyzed cell through the unique rearranged sequence of its antigen receptor (BCR or TCR, respectively) and link it to the functional state inferred from transcriptome analysis. Here we introduce FB5P-seq, a FACS-based 5'-end scRNA-seq method for cost-effective, integrative analysis of transcriptome and paired BCR or TCR repertoire in phenotypically defined B and T cell subsets. We describe in detail the experimental workflow and provide a robust bioinformatics pipeline for computing gene count matrices and reconstructing repertoire sequences from FB5P-seq data. We further present two applications of FB5P-seq for the analysis of human tonsil B cell subsets and peripheral blood antigen-specific CD4 T cells. We believe that our novel integrative scRNA-seq method will be a valuable option to study rare adaptive immune cell subsets in immunology research.

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Single cell five-prime end sequencing of PBMC - resting and stimulated

Project description:Single cell five-prime end sequencing of PBMC - resting and stimulated. Used to asses the five-prime sequencing method in the detection of cis-regulatory elements using SCAFE (see publication)

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Project description:Similar with others, our data proved that antigen-specific CD8+ T cells from mice primed with DNA and boosted by VACV were much more sensitive to antigen stimulation than those from DNA-boost. Since the mechanisms of in vivo tuning of antigen sensitivity (also termed functional avidity) is still not defined, we compared this two vaccination regimen at gene expression level. Results provide important information of which genes were selectively activated by VACV boost vaccination. For example, data shows that the expression levels of genes involved in Cancer and Wnt signaling pathways is more higher in DNA prime-VACV boost regimen that DNA prime-DNA boost vaccination. To obtain sufficient of antigen-specific cells for microarray analysis, the OVA-specific CD8+ T cells from OT-1 mice were adoptively transferred into wild type mice and then immunized by DNA and VACV vaccine encoding OVA. Four week later, mice were scarificed and antigen-specific CD8+ T cells were emriched by CD45.1-PE antibody and anti-PE MicroBeads from splenocytes.Total RNA was extracted by the RNeasy Mini Kit (QIAGEN, Germany). Followed by amplification and biotin labeling, the samples were hybridized using Illumina Total Prep RNA Amplification Kit (Ambion, USA). Mouse WG-6v2 Expression BeadChips were used for analysis of transcriptome.

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