High-throughput RNA Isoform Sequencing using Programmable cDNA Concatenation
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PROVIDER: PRJNA933950 | ENA |
REPOSITORIES: ENA
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Project description:High-throughput RNA Isoform Sequencing using Programmable cDNA Concatenation
| PRJNA933949 | ENA
Project description:mRNA isoform detection by long-read sequencing of 10x single cell cDNA libraries
| PRJEB85328 | ENA
Project description:Through alternative splicing, most human genes express multiple isoforms that may have distinct or even antagonistic functions. To infer isoform regulation based on data from high-throughput sequencing of cDNA fragments (RNA-Seq), we have developed MISO, a computational model that estimates the expression level of alternatively spliced exons and mRNA isoforms and provides intuitive measures of confidence in these estimates. Incorporation of the length distribution of inserted cDNA fragments in paired-end RNA-Seq analysis in MISO enables dramatic improvements in estimation of alternative splicing levels relative to previous methods. We show that one lane of paired-end RNA-Seq data can provide far more information about splicing than two lanes of single-end data, depending critically on properties of the distribution of cDNA fragment lengths in the sequenced library. MISO also leads to an intuitive method to detect differentially regulated exons or isoforms. Application of this method implicates the RNA splicing factor hnRNP H in regulation of alternative cleavage and polyadenylation, a role that is supported by UV crosslinking/immunoprecipitation/high-throughput sequencing (CLIP-Seq) analysis. Together, our results provide a probabilistic framework for RNA-Seq analysis, derive functional insights into pre-mRNA processing, and yield guidelines for the optimal design of RNA-Seq experiments for studies of gene and isoform expression.
2010-11-07 | GSE23694 | GEO
Project description:Purpose: Mutations in TP53 induce autoantibody immune responses in a subset of cancer patients, which have been proposed as biomarkers for early detection. Here, we investigate the association of p53 specific autoantibodies with multiple tumor subtypes and determine the association with p53 mutation status and epitope specificity. Experimental Design: IgG p53 autoantibodies (p53-AAb), were quantified in 412 serum saples using a programmable ELISA assay from patients with serous ovarian, pancreatic adenocarcinoma, and breast cancer. To determine if patients generated mutation specific autoantibodies we designed a panel of the most relevant 51 p53 point mutant proteins, to be displayed on custom programmable protein microarrays. To determine the epitope specificity we displayed 12 overlapping tiling fragments and 38 N- and C-terminal deletions spanning the length of the wild-type p53 proteins. Results: We detected p53-AAb with sensitivities of 58.8% (ovarian), 22% (pancreatic), 32% (triple negative breast cancer), and 10.2% (HER2+ breast cancer) at 94% specificity. Sera with p53-AAb contained broadly-reactive autoantibodies to 51 displayed p53 mutant proteins, demonstrating a polyclonal response to common epitopes. All p53-AAb displayed broad polyclonal immune response to both continuous and discontinuous epitopes at the N- and C-terminus as well as the DNA binding domain. Conclusion and clinical relevance: In this comprehensive analysis, mutations in tumor p53 induce strong, polyclonal autoantibodies with broadly reactive epitope specificity. The immunoreactivity was compared between 60 pancreactic ductal adenocarcinoma cases and 63 benign pancreatic disease controls against 52 unique mutant p53 and 379 human proteins that were printed on microscope slides. [Contributor] Arizona State University
2016-06-01 | E-GEOD-77949 | biostudies-arrayexpress
Project description:Although long-read single-cell RNA isoform sequencing (scISO-Seq) can reveal alternative RNA splicing in individual cells, it suffers from a low read throughput. Here, we introduce HIT-scISOseq, a method that removes most artifact cDNAs and concatenates multiple cDNAs for PacBio circular consensus sequencing (CCS) to achieve high-throughput and high-accuracy single-cell RNA isoform sequencing. HIT-scISOseq can yield >10 million high-accuracy long-reads in a single PacBio Sequel II SMRT Cell 8M. We also report the development of scISA-Tools that demultiplex HIT-scISOseq concatenated reads into single-cell cDNA reads with >99.99% accuracy and specificity. We apply HIT-scISOseq to characterize the transcriptomes of 3375 corneal limbus cells and reveal cell-type-specific isoform expression in them. HIT-scISOseq is a high-throughput, high-accuracy, technically accessible method and it can accelerate the burgeoning field of long-read single-cell transcriptomics.
| S-EPMC10164132 | biostudies-literature
Project description:Through alternative splicing, most human genes express multiple isoforms that may have distinct or even antagonistic functions. To infer isoform regulation based on data from high-throughput sequencing of cDNA fragments (RNA-Seq), we have developed MISO, a computational model that estimates the expression level of alternatively spliced exons and mRNA isoforms and provides intuitive measures of con?dence in these estimates. Incorporation of the length distribution of inserted cDNA fragments in paired-end RNA-Seq analysis in MISO enables dramatic improvements in estimation of alternative splicing levels relative to previous methods. We show that one lane of paired-end RNA-Seq data can provide far more information about splicing than two lanes of single-end data, depending critically on properties of the distribution of cDNA fragment lengths in the sequenced library. MISO also leads to an intuitive method to detect di?erentially regulated exons or isoforms. Application of this method implicates the RNA splicing factor hnRNP H in regulation of alternative cleavage and polyadenylation, a role that is supported by UV crosslinking/immunoprecipitation/high-throughput sequencing (CLIP-Seq) analysis. Together, our results provide a probabilistic framework for RNA-Seq analysis, derive functional insights into pre-mRNA processing, and yield guidelines for the optimal design of RNA-Seq experiments for studies of gene and isoform expression. CLIPseq of hnRNP H in HEK 293T cells. RNAseq of polyA+ RNA from C2C12 mouse myoblasts stably expressing an empty vector or a vector containing an shRNA against CUGBP1. Libraries of two different insert lengths were created and examined.
2010-11-07 | E-GEOD-23694 | biostudies-arrayexpress
Project description:Recent studies claim that N4-acetylcytidine (ac4C) modification of RNA confers crucial regulatory roles, such as increasing translation efficiency and prolonging its half-life. However, the absence of methods for selectively acetylating specific RNA molecules hampers linking ac4C to cell physiology. Here, we developed an efficient molecular tool that incorporates ac4C on a specific transcript of interest. Through protein engineering, we developed a hyperactive variant of N-acetyltransferase 10 (NAT10), designated eNAT10. When fused to the programmable RNA-targeting protein dCas13, eNAT10 enables robust acetylation of various target RNAs in multiple contexts. RNA acetylation by dCas13-eNAT10 was highly dependent on co-transfected guide RNA, highlighting its specificity. We also describe the first programmable RNA chemical modification in vivo using dual-AAV. Utilizing our system, we found that acetylation of RNA may modulate the subcellular localization of modified transcripts. We anticipate that our tool will facilitate numerous studies on ac4C functions across different cellular and disease contexts.
2025-04-08 | GSE293920 | GEO
Project description:Programmable RNA acetylation with CRISPR-Cas13
| PRJNA1247425 | ENA
Project description:Cells utilize ubiquitin as a posttranslational protein modifier to convey various signals such as proteasomal degradation. The dysfunction of ubiquitylation or following proteasomal degradation can give rise to the accumulation and aggregation of improperly ubiquitylated proteins, which is known to be a general causation of many neurodegenerative diseases. Thus, the characterization of substrate peptide sequences of E3 ligases is crucial in biological and pharmaceutical sciences. In this study, we developed a novel high-throughput screening system for substrate peptide sequences of E3 ligases using a cDNA display method, which enables covalent conjugation between peptide sequences and their corresponding cDNA sequences. First, we focused on the MDM2 E3 ligase and its known peptide substrate as a model to establish the screening method, and confirmed that cDNA display method was compatible with in vitro ubiquitylation. Then, we demonstrated identification of MDM2 substrate sequences from random libraries to identify a novel motif (VKFTGGQLA). Bioinformatics analysis of the hit sequences was performed to gain insight about endogenous substrate proteins.
| S-EPMC11664913 | biostudies-literature