Preparation of mouse pancreatic tumor for single-cell RNA sequencing and analysis of the data

Surumbayeva, Aizhan; Kotliar, Michael; Gabitova-Cornell, Linara; Kartashov, Andrey V.; Peri, Suraj; Salomonis, Nathan; Barski, Artem; Astsaturov, Igor

doi:10.1016/j.xpro.2021.100989

Cited by 5 publications

(4 citation statements)

References 9 publications

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“…The analysis of sc sequencing data includes several stages and can be roughly divided into preprocessing that assigns reads to cells and genes or peaks, low-quality cell removal, dimensionality reduction, clustering, and cell annotation steps [32, 33]. Each of these can be accomplished by running one or several pipelines designed for a specific sequencing data type (scRNA-Seq, scATAC-Seq or scMultiome).…”

Section: Methodsmentioning

confidence: 99%

Accelerating Single-Cell Sequencing Data Analysis with SciDAP: A User-Friendly Approach

Kotliar,

Kartashov,

Barski

2024

Preprint

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Single-cell (sc) RNA, ATAC and Multiome sequencing became powerful tools for uncovering biological and disease mechanisms. Unfortunately, manual analysis of sc data presents multiple challenges due to large data volumes and complexity of configuration parameters. This complexity, as well as not being able to reproduce a computational environment, affects the reproducibility of analysis results. The Scientific Data Analysis Platform (https://SciDAP.com) allows biologists without computational expertise to analyze sequencing-based data using portable and reproducible pipelines written in Common Workflow Language (CWL). Our suite of computational pipelines addresses the most common needs in scRNA-Seq, scATAC-Seq and scMultiome data analysis. When executed on SciDAP, it offers a user-friendly alternative to manual data processing, eliminating the need for coding expertise. In this protocol, we describe the use of SciDAP to analyze scMultiome data. Similar approaches can be used for analysis of scRNA-Seq, scATAC-Seq and scVDJ-Seq datasets.

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Section: Methodsmentioning

confidence: 99%

Accelerating Single-Cell Sequencing Data Analysis with SciDAP: A User-Friendly Approach

Kotliar,

Kartashov,

Barski

2024

Preprint

Self Cite

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“…Afterwards, red blood cells were lysed with ACK Lysing buffer for 5 min, washed again in staining buffer and cells proceeded to staining for flow cytometry analysis. For single cell RNA sequencing analysis, the suspensions were subjected to an extra step for dead cell removal using (Dead Cell Removal Kit, Miltenyi Biotec) (207).…”

Section: Single Cell Suspensions From Pancreatic Tumorsmentioning

confidence: 99%

Netrin G1 Ligand is a new stromal immunomodulator that promotes pancreatic cancer

Vendramini-Costa,

Francescone,

Franco-Barraza

et al. 2024

Preprint

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Understanding pancreatic cancer biology is fundamental for identifying new targets and for developing more effective therapies. In particular, the contribution of the stromal microenvironment to pancreatic cancer tumorigenesis requires further exploration. Here, we report the stromal roles of the synaptic protein Netrin G1 Ligand (NGL-1) in pancreatic cancer, uncovering its pro-tumor functions in cancer-associated fibroblasts and in immune cells. We observed that the stromal expression of NGL-1 inversely correlated with patient overall survival. Moreover, germline knockout (KO) mice for NGL-1 presented decreased tumor burden, with a microenvironment that is less supportive of tumor growth. Of note, tumors from NGL-1 KO mice produced less immunosuppressive cytokines and displayed an increased percentage of CD8+ T cells than those from control mice, while preserving the physical structure of the tumor microenvironment. These effects were shown to be mediated by NGL-1 in both immune cells and in the local stroma, in a TGF-β-dependent manner. While myeloid cells lacking NGL-1 decreased the production of immunosuppressive cytokines, NGL-1 KO T cells showed increased proliferation rates and overall polyfunctionality compared to control T cells. CAFs lacking NGL-1 were less immunosuppressive than controls, with overall decreased production of pro-tumor cytokines and compromised ability to inhibit CD8+ T cells activation. Mechanistically, these CAFs downregulated components of the TGF-β pathway, AP-1 and NFAT transcription factor families, resulting in a less tumor-supportive phenotype. Finally, targeting NGL-1 genetically or using a functionally antagonistic small peptide phenocopied the effects of chemotherapy, while modulating the immunosuppressive tumor microenvironment (TME), rather than eliminating it. We propose NGL-1 as a new local stroma and immunomodulatory molecule, with pro-tumor roles in pancreatic cancer.

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“…Preprocessing Bioinformatics analysis of single-cell RNA sequencing data was conducted as described previsoly 76 Low quality cell removal Low quality cells were removed in two iterations. First, the following filtering thresholds were applied per cell -from 80 to 6000 genes, minimum 500 uniquely mapped fragments, maximum 5 percent of transcripts mapped to mitochondrial genes.…”

Section: Single Cell Rna-sequencing (Scrna-seq) Bioinformatic Pipelinementioning

confidence: 99%

Caspase 8 protects pancreatic neoplasia from KRAS-driven necroptotic priming

Tishina,

Dahlhaus,

Androulidaki

et al. 2023

Preprint

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Caspase 8 regulates normal tissue homeostasis by executing apoptosis and protecting from necroptosis. Several aggressive cancers express high levels of caspase 8, yet its function in neoplastic disease remains poorly explored. Here, we find that oncogenic KRAS-driven neoplastic transformation induces a transcriptional state of necroptotic priming, but necroptosis itself is kept in check by co-upregulation of caspase 8. Mice in which caspase 8 is deleted in the pancreas manifested a drastic decrease in precursor lesion formation in a KRASG12D-driven model of pancreatic cancer. Co-deletion of the essential necroptosis effector MLKL reversed the protective effects of caspase 8 ablation, and promoted metastasis. Mechanistically, oncogenic KRAS induced a STING-dependent type I interferon response, resulting in upregulation of necroptosis pathway components. High caspase 8 expression in precursor lesions was a result of co-selection to prevent necroptosis. Therefore, triggering necroptosis by blocking caspase 8 activity was therapeutically highly efficacious in models of genetically engineered PDAC in vivo. These results assign a tumor-protective function to caspase 8 in PDAC and show that overcoming caspase 8 activity has therapeutic benefit in this incurable malignancy.

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Preparation of mouse pancreatic tumor for single-cell RNA sequencing and analysis of the data

Cited by 5 publications

References 9 publications

Accelerating Single-Cell Sequencing Data Analysis with SciDAP: A User-Friendly Approach

Accelerating Single-Cell Sequencing Data Analysis with SciDAP: A User-Friendly Approach

Netrin G1 Ligand is a new stromal immunomodulator that promotes pancreatic cancer

Caspase 8 protects pancreatic neoplasia from KRAS-driven necroptotic priming

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