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CLEAR: Coverage-based Limiting-cell Experiment Analysis for RNA-seq (mouse)

ABSTRACT: Direct cDNA preamplification protocols developed for single-cell RNA-seq (scRNA-seq) have enabled transcriptome profiling of rare cells without having to pool multiple samples or to perform RNA extraction. We term this approach limiting-cell RNA-seq (lcRNA-seq). Unlike scRNA-seq, which focuses on ‘cell-atlasing’, lcRNA-seq focuses on identifying differentially expressed genes (DEGs) between experimental groups. This requires accounting for systems noise which can obscure biological differences. We present CLEAR, a workflow that identifies robust transcripts in lcRNA-seq data for between-group comparisons. To develop CLEAR, we compared DEGs from FACS-derived CD5+ and CD5- cells from a chronic lymphocytic leukemia patient at different input RNA levels. When using CLEAR transcripts vs. using all available transcripts, downstream analyses reveal more consistent DEGs, improved Principal Component Analysis separation by cell type, and consistency across input RNA amounts. CLEAR also performs well on external sc- and lcRNA-seq data and an internal murine neural cell lcRNA-seq data set.

ORGANISM(S): Mus musculus

PROVIDER: GSE115033 | GEO | 2020/02/18

REPOSITORIES: GEO

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CLEAR: Coverage-based Limiting-cell Experiment Analysis for RNA-seq (human)

Project description:Direct cDNA preamplification protocols developed for single-cell RNA-seq (scRNA-seq) have enabled transcriptome profiling of rare cells without having to pool multiple samples or to perform RNA extraction. We term this approach limiting-cell RNA-seq (lcRNA-seq). Unlike scRNA-seq, which focuses on ‘cell-atlasing’, lcRNA-seq focuses on identifying differentially expressed genes (DEGs) between experimental groups. This requires accounting for systems noise which can obscure biological differences. We present CLEAR, a workflow that identifies robust transcripts in lcRNA-seq data for between-group comparisons. To develop CLEAR, we compared DEGs from FACS-derived CD5+ and CD5- cells from a chronic lymphocytic leukemia patient at different input RNA levels. When using CLEAR transcripts vs. using all available transcripts, downstream analyses reveal more consistent DEGs, improved Principal Component Analysis separation by cell type, and consistency across input RNA amounts. CLEAR also performs well on external sc- and lcRNA-seq data and an internal murine neural cell lcRNA-seq data set.

2020-02-18 | GSE115032 | GEO

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Human HepG2 ITRAQ LC-MS/MS - Integrated proteomic and transcriptomic analysis revealslong noncoding RNA HOTAIR promotes hepatocellular carcinoma cell proliferation by regulating opioid growth factor receptor (OGFr)

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RNA Sequencing Analysis of Gene Expression Profiles in Cic-null B-1a and Follicular B (Fo B) cells

Project description:Purpose: The goal of this study is to compare and examine the transcriptional profiles in control versus Cic-deficient B cells, specifically splenic B-1a and Fo B cells, by mRNA sequencing. Methods: Splenic B-1a (IgM+, CD19+, CD5+, CD43+) and Fo B (B220+, CD93-, CD23+, CD21lo-mid) cells were sorted by a MoFlo-XDP (Beckman Coulter). Total RNA was extracted using TRIzol Reagent (GeneAll), according to the manufacturer’s instructions. The DNA library for mRNA sequencing was generated using SMART-seq v4 Ultra Input RNA Kit (Clontech, for B-1a cells) and TruSeq RNA Sample Prep Kit v2 (Illumina, for Fo B cells). DNA libraries were multiplexed and pooled for sequencing on the Illumina Hiseq 4000 platform. Results: Trimmed reads were mapped to mouse reference genome (mm10 RefSeq) with HISAT2, a splice-aware aligner, and transcripts were assembled by StringTie with aligned reads, for known transcripts, novel transcripts, and alternatively spliced transcripts. A total of 712 genes were differentially expressed in Cic-null B-1a cells (325 upregulated and 387 downregulated), while 357 genes were differentially expressed in Cic-deficient Fo B cells (180 upregulated and 178 downregulated) when compared with their respective control cells (fold change >1.2 and P < 0.05). Several known CIC target genes, including Etv1, Etv4, Etv5, Spry4, Spred1, Spred2, Dusp4, Dusp6, and Per2, were included in the list of the upregulated differentially expressed genes (DEGs) in either Cic-null B-1a or Fo B cells or both, validating the reliability of RNA sequencing results. Conclusions: Our study presents the first comparative gene expression analysis of B-1a and Fo B cells from control and B cell-specific Cic-deficient mice. We concluded that CIC deficiency promotes B-1a cell development through enhancing the BCR signaling pathway. The data reported here also provide references for the gene expression profiles of murine B-1a and Fo B cells.

2021-12-16 | GSE163455 | GEO

Acupuncture potentiates anti-PD-1 efficacy by promoting CD5+ dentritic cells and T cell-mediated tumor immunity in breast cancer

Project description:Acupuncture has been considered as an available complement therapy against various malignancy cancer by regulating immunity. However, it is not clear whether acupuncture influences the antitumor immune efficacy of PD-1 inhibitor. This study evaluates the efficacy of acupuncture combined with PD-1 inhibitor on the antitumor immune response against breast cancer and revealed the potential molecular mechanisms based on RNA-Seq transcriptome analysis. The results depicted that acupuncture combined with PD-1 inhibitor significantly hindered tumor development by inducing tumor cell apoptosis and inhibiting tumor growth, angiogenesis and metastasis. RNA-Seq analysis indicated that the different expression genes upon acupuncture intervention mainly enriched in immune response, T cell activation and cytokine interaction etc. items containing immune cell-related CD genes (CD5, CD4 and CD8 etc.). Acupuncture stimulation enhanced CD5 expression that had a positive correlation with overall survival of breast cancer patients. The improved immunity including the enhancement of CD5+ dendritic cell, CD4+ and CD8+ T cell content as well as various cytokine (IL-2, IL-6, TNF-α, IFN-γ) secretion in serum was found upon acupuncture combination with PD-1 inhibitor treatment. Taken together, present work reveals that acupuncture intervention promotes the antitumor immune response of PD-1 inhibitor, providing a prospective therapy for ICI therapy against breast cancer.

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3ff3c74c-d0b2-4c2b-aa48-c534cd259731 - samples

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