Project description:4 groups of mice : Control, antibiotics, antibiotics + 4days of recolonization, antibiotics + 4days of recolonization + Enterocloster clostridioformis. Tumor draining lymph node were harvested after CFSE injection were harvested and CFSE+ cells were sorted and proccessed in order to generate single cell RNA-sequencing using BD Rhapsody mouse immune response targeted panel. Groups were barcoded using BD Rhapsody Multiplexing Kit.
Project description:To investigate the role of aging in the regulation of ILC3 development and function, we used aged mice and carried out single-cell RNA sequencing (BD Rhapsody) on aged gut ILC3s.
Project description:To investigate the role of ThPOK in the regulation of ILC3 development and function, we established conventional ThPOK knockout mice and carried out single-cell RNA sequencing (BD Rhapsody) on intestinal ILC3s.
Project description:To investigate the role of Cxxc1 in the regulation of ILC3 development and function, we used Cxxc1flox/floxRorccre mice and carried out single-cell RNA sequencing (BD Rhapsody) on small intestinal ILC3s.
Project description:To investigate the role of aging in the regulation of ILC3 development and function, we used the young mice as control and carried out single-cell RNA sequencing (BD Rhapsody) on young gut ILC3s.
Project description:KLB-Cre mice were crossed to Ai14 mice to label KLB-expressing cells with tdTomato. The basolateral amygdala was then dissected out and tdTomato positive cells were isolated using FACs sorting. FACs sorted cells were then captured for single cell RNA sequencing analysis using the BD Rhapsody Whole Transcriptome Analysis kit.
Project description:We used targeted single cell RNA sequencing using a gene set composed of the BD Rhapsody Human Immune Response Panel, TCR panel, and a custom panel of 61 malignancy-associated genes and T/B cell receptor sequencing to characterize single cell populations in hypopigmented mycosis fungoides (HMF). Using a reference mapping model built on publically deposited classic mycosis fungoides single cell RNA sequencing datasets, we identified a predicted malignant cell population independent from the clonal T cell population in HMF.
Project description:Immunotherapy using CD19-directed chimeric antigen receptor (CAR)-T cells has shown excellent results for treatment of B-cell leukaemia and lymphoma. To produce CAR-T cells, the patient’s own T cells are isolated from the blood and modified in a laboratory with a genetic vector to express a tumor antigen-directed CAR on its surface. The CAR-T cells are then expanded in numbers and given back to the patient with the aim to eradicate the tumors. However, some patients display primary resistance to CAR-T treatment while others relapse quickly after CAR-T treatment. In this experiment, we seek to understand whether the quality of the individual CAR-T cell product the patients were given can predict outcome to the therapy. We investigate the transcriptional profile of the individual CAR-T infusion products using single-cell RNA sequencing. In this dataset, we identified a T cell subset correlating with response that could be used as an indicator for clinical outcome. Targeted RNA and protein single-cell libraries were obtained using the BD Rhapsody platform (BD Biosciences). In total four separate targeted libraries were produced with 6 patients per library. Sequencing was performed on NovaSeq 6000 S1 sequencer at the SNP&SEQ Technology Platform (Uppsala, Sweden). The raw scRNA-seq data was pre-processed by BD Biosciences using the Rhapsody Analysis pipeline to convert the raw reads into Unique Molecular Identifier (UMI) counts. UMIs are further adjusted within Rhapsody by applying BD’s Recursive Substitution Error Correction (RSEC) and Distribution-Based Error Correction (DBEC) in order to remove false UMIs caused by sequencing or library preparation errors. Pooled samples were deconvoluted using Sample-tag reads. The scRNA-seq and AbSeq counts were loaded, processed and used for clustering and differential gene expression with Seurat v. 4.0.0.
Project description:Single-cell transcriptomics has emerged as the preferred tool to define cell identity through the analysis of gene expression signatures. However, there are limited studies that have comprehensively compared the performance of different scRNAseq systems in complex tissues. Here, we present a systematic comparison of three well-established high throughput 3’-scRNAseq platforms: Drop-seq, 10x Chromium and BD Rhapsody; using tumours that present high cell diversity. Our experimental design includes both fresh and artificially damaged samples from the same tumours, which also provides a comparable dataset to examine their performance under challenging conditions. The performance metrics used in this study consist of gene sensitivity, mitochondrial content, reproducibility, clustering capabilities, cell type representation and ambient RNA contamination. These analyses showed that BD Rhapsody and 10x Chromium have similar but higher gene sensitivity than Drop-seq, while BD Rhapsody has the highest mitochondrial content. Interestingly, we found cell type detection biases between platforms, including a lower proportion of endothelial and myofibroblast cells in BD Rhapsody and lower gene sensitivity in granulocytes for 10x Chromium. Moreover, the source of the ambient noise was different between plate-based and droplet-based platforms. In conclusion, our reported platform differential performance should be considered for the selection of the scRNAseq method during the study experimental designs.
