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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

Systematic evaluation of RNA-Seq preparation protocol performance (RNASeq: SMARTer)

Project description:In this study, we present a comprehensive evaluation of four RNA-Seq library preparation methods. We used three standard input protocols, the Illumina TruSeq Stranded Total RNA and TruSeq Stranded mRNA kits, and a modified NuGEN Ovation v2 kit; and an ultra-low-input RNA protocol, the TaKaRa SMARTer Ultra Low RNA Kit v3. Our evaluation of these kits included quality control measures such as overall reproducibility, 5’ and 3’ end-bias, and the identification of DEGs, lncRNAs, and alternatively spliced transcripts. Overall, we found that the two Illumina kits were most similar in terms of recovering DEGs, and the Illumina, modified NuGEN, and TaKaRa kits allowed identification of a similar set of DEGs. However, we also discovered that the Illumina, NuGEN and TaKaRa kits each enriched for different sets of genes.

2019-06-27 | GSE131396 | GEO

Systematic evaluation of RNA-Seq preparation protocol performance (RNASeq: TruSeq)

2019-06-27 | GSE131397 | GEO

Transcriptional Profiling of Psoriasis Using RNA-seq Reveals Previously Unidentified Differentially Expressed genes

Project description:The transcriptomic profiling of psoriasis has led to an increased understanding of disease pathogenesis. Although microarray technologies have been instrumental in this regard, it is clear that these tools detect an incomplete set of DEGs. RNA-seq can be used to supplement these prior technologies. Here, the use of RNAseq methods substantially increased the number of psoriasis-related DEGs. Furthermore, DEGs that were uniquely identified by RNA-seq, but not in other published microarray studies, further supported the role of IL-17 and tumor necrosis factor-a synergy in psoriasis. Examination of one of these factors at the protein level confirmed that RNA-seq is a powerful tool that can be used to identify molecular factors present in psoriasis lesions, and may be useful in the identification of therapeutic targets that to our knowledge have not been reported previously. Further studies are in progress to determine the biological significance of DEGs uniquely discovered by RNA-seq. To define the transcriptomic profile of psoriatic skin, three pairs of lesional and nonlesional skin biopsy specimens were taken from patients with untreated moderate-to-severe plaque psoriasis.

2012-10-22 | E-GEOD-41745 | biostudies-arrayexpress

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)

Project description:Long non-coding RNA HOX transcript antisense RNA (HOTAIR) is involved in human tumorigenesis and dysregulated in hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying HOTAIR functions in HCC are not well understood. Here, an integrated RNA sequencing transcriptomic and quantitative proteomic analysis were employed to systematically explore the regulatory role of HOTAIR in HCC. A total of 673 transcripts and 298 proteins were identified to be dysregulated after HOTAIR inhibition. Bioinformatics studies indicated that the differentially expressed genes (DEGs) and proteins (DEPs) are involved in many biological processes, especially cancer-related signalling pathways.

2017-11-24 | PXD006554 | Pride

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.

2024-06-03 | GSE268566 | GEO

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

Project description:Human data for transcriptome (bulk RNA-Seq) in eight B-cell precursors: HSC, CLP, pro-B, pre-B, Immature B, Transitional B, Naive B CD5-, Naive B CD5+ cells

| EGAD00001010906 | EGA

H2S regulation the metabolism of cucumber in response to salt-stress through transcriptome and proteome analysis

Project description:In a previous study, we found that H2S alleviates salinity stress in cucumber by maintaining the Na+/K+ balance and by regulating H2S metabolism and the oxidative stress response. However, little is known about the molecular mechanisms behind H2S-regulated salt-stress tolerance in cucumber. Here, an integrated transcriptomic and proteomic analysis based on RNA-seq and 2-DE was used to investigate the global mechanism underlying H2S-regulated salt-stress tolerance. In total, 11 761 differentially expressed genes (DEGs) and 61 differentially expressed proteins (DEPs) were identified. Analysis of the pathways associated with the DEGs showed that salt stress enriched expression of genes in primary and energy metabolism, such as photosynthesis, carbon metabolism and biosynthesis of amino acids. Application of H2S significantly decreased these DEGs but enriched DEGs related to plant-pathogen interaction, sulfur-containing metabolism, cell defense and signal transduction pathways. Notably, changes related to sulfur-containing metabolism and cell defense were also observed through proteome analysis, such as Cysteine synthase 1, Glutathione S-transferase U25-like, Protein disulfide-isomerase and Peroxidase 2. We present the first global analysis of the mechanism underlying H2S regulation of salt-stress tolerance in cucumber through tracking changes in the expression of specific proteins and genes.

2021-09-09 | PXD020642 | Pride

Targeted RNA sequencing enhances gene expression profiling of ultra-low input samples

Project description:RNA-seq is the standard method for profiling gene expression in many biological systems. Due to the wide dynamic range and complex nature of the transcriptome, RNA-seq provides an incomplete characterisation, especially of lowly expressed genes and transcripts. Targeted RNA sequencing (RNA CaptureSeq) focuses sequencing on genes of interest, providing exquisite sensitivity for transcript detection and quantification. However, uses of CaptureSeq have focused on bulk samples and its performance on very small populations of cells is unknown. Here we show CaptureSeq greatly enhances transcriptomic profiling of target genes in ultra-low-input samples and provides equivalent performance to that on bulk samples. We validate the performance of CaptureSeq using multiple probe sets on samples of iPSC-derived cortical neurons. We demonstrate up to 275-fold enrichment for target genes, the detection of 10% additional genes and a greater than 5-fold increase in identified gene isoforms. Analysis of spike-in controls demonstrated CaptureSeq improved both detection sensitivity and expression quantification. Comparison to the CORTECON database of cerebral cortex development revealed CaptureSeq enhanced the identification of sample differentiation stage. CaptureSeq provides sensitive, reliable and quantitative expression measurements on hundreds-to-thousands of target genes from ultra-low-input samples and has the potential to greatly enhance transcriptomic profiling when samples are limiting.

2019-11-16 | GSE140442 | GEO

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