Ontology highlight
ABSTRACT:
PROVIDER: PRJNA902089 | ENA |
REPOSITORIES: ENA
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Project description:PacBio long-read sequencing of the tprK gene
| PRJNA902035 | ENA
Project description:Objectives: To perform long-read transcriptome and proteome profiling of pathogen-stimulated peripheral blood mononuclear cells (PBMCs) from healthy donors. We aim to discover new transcripts and protein isoforms expressed during immune responses to diverse pathogens. Methods: PBMCs were exposed to four microbial stimuli for 24 hours: the TLR4 ligand lipopolysaccharide (LPS), the TLR3 ligand Poly(I:C), heat-inactivated Staphylococcus aureus, Candida albicans, and RPMI medium as negative controls. Long-read sequencing (PacBio) of one donor and secretome proteomics and short-read sequencing of five donors were performed. IsoQuant was used for transcriptome construction, Metamorpheus/FlashLFQ for proteome analysis, and Illumina short-read 3’-end mRNA sequencing for transcript quantification. Results: Long-read transcriptome profiling reveals the expression of novel sequences and isoform switching induced upon pathogen stimulation, including transcripts that are difficult to detect using traditional short-read sequencing. We observe widespread loss of intron retention as a common result of all pathogen stimulations. We highlight novel transcripts of NFKB1 and CASP1 that may indicate novel immunological mechanisms. In general, RNA expression differences did not result in differences in the amounts of secreted proteins. Interindividual differences in the proteome were larger than the differences between stimulated and unstimulated PBMCs. Clustering analysis of secreted proteins revealed a correlation between chemokine (receptor) expression on the RNA and protein levels in C. albicans- and Poly(I:C)-stimulated PBMCs. Conclusion: Isoform aware long-read sequencing of pathogen-stimulated immune cells highlights the potential of these methods to identify novel transcripts, revealing a more complex transcriptome landscape than previously appreciated.
2023-09-16 | PXD045237 | Pride
Project description:Here we describe CapTrap-Seq, an experimental workflow designed to address the problem of reduced transcript end detection by long-read RNA sequencing methods, especially at the 5' ends. We apply CapTrap-Seq to profile transcriptomes of the human heart and brain and we compared the obtained results with other library preparation approaches. CapTrap-Seq is a platform-agnostic method and here tested the method by using 3 different long-read sequencing platforms: MinION (ONT), Sequel (PacBaio) and Sequel II (PacBio).
2023-12-20 | E-MTAB-13063 | biostudies-arrayexpress
Project description:Grass pea seeds and seedlings protein extracts were chromatographically fractionated. To identify the β-ODAP synthase enzyme, active fractions, as determined by a colorimetric assay that detects the presence of a derivative of free L-α,β-diaminopropionic acid (L-DAPA), were subjected to tryptic digestion and LC-MS/MS and searched against a database containing translated sequences from a long-read PacBio mRNA sequencing of grass pea seeds and seedlings.
2022-05-19 | PXD030847 | Pride
Project description:Long-read sequencing technologies such as Iso-Seq (PacBio Inc.) generate highly accurate sequences of full-length mRNA transcript isoforms. Long-read transcriptomics may be especially useful in the context of lymphocyte functional plasticity as it relates to human health and disease. However, no long-read isoform-aware reference transcriptomes of human circulating lymphocytes seem to be publicly available despite being valuable as benchmarks in a variety of transcriptomic studies. To begin to fill this gap, we purified four lymphocyte subsets (CD4 T, CD8 T, NK, and Pan B cells) from the peripheral blood of a healthy male donor and obtained high-quality RNA (RIN>8) for PacBio Iso-Seq analysis and parallel RNA-Seq analysis.
2022-09-16 | GSE202328 | GEO
Project description:Long-read sequencing technologies such as Iso-Seq (PacBio Inc.) generate highly accurate sequences of full-length mRNA transcript isoforms. Long-read transcriptomics may be especially useful in the context of lymphocyte functional plasticity as it relates to human health and disease. However, no long-read isoform-aware reference transcriptomes of human circulating lymphocytes seem to be publicly available despite being valuable as benchmarks in a variety of transcriptomic studies. To begin to fill this gap, we purified four lymphocyte subsets (CD4 T, CD8 T, NK, and Pan B cells) from the peripheral blood of a healthy male donor and obtained high-quality RNA (RIN>8) for PacBio Iso-Seq analysis and parallel RNA-Seq analysis.
2022-09-16 | GSE202327 | GEO
Project description:STAMBPL1 plays a previously unappreciated role in post-transcriptional regulation, especially in RNA splicing pathways. Thus, PacBio long-read iso-sequencing was performed to acquire a high-precision transcription landscape after STAMBPL1 knockingdown in Huh7 cells
2024-09-11 | GSE276508 | GEO
Project description:The steady state expression of each gene is the result of transcriptional rate, dynamic processing, and degradation of RNA. While regular RNA-seq methods only measure steady state expression levels, RNA-seq of metabolically labeled RNA identifies transcripts that were transcribed during the window of metabolic labeling. Whereas short-read RNA sequencing can identify metabolically labeled RNA at the gene level, long-read sequencing results in dramatically improved resolution of isoform-level transcription. Here we combine thiouridine-to-cytosine conversion (TUC) with PacBio long-read sequencing to study the dynamics of mRNA transcription in the GM12878 cell line. We show that using long-TUC-seq, we can detect metabolically labeled mRNA of distinct isoforms more reliably than using short reads. Long-TUC-seq holds the promise of capturing isoform dynamics robustly and without the need for enrichment.
2020-04-30 | GSE149551 | GEO
Project description:The human neural retina is enriched for alternative splicing, and it is estimated that more than 10% of variants associated with inherited retinal diseases (IRDs) alter splicing. Previous research mainly used short-read RNA-sequencing techniques to investigate retina-specific splicing and splicing factors. However, this technique provides limited information about transcript isoforms. To gain a deeper understanding of the human neural retina and its isoforms, we generated a proteogenomic atlas that combined PacBio long-read RNA-sequencing data with mass-spectrometry and whole-genome sequencing data from three healthy human neural retina samples. RNA-sequencing revealed that one-third of all transcripts were novel, and for IRD-associated genes, even 43% were novel. The most common novel elements of these transcripts were alternative poly(A) sites, exon elongation, and intron retention. Some novel elements affect the non-coding region but for more than 50% of the novel transcripts a novel open reading frame was predicted. Using proteomics, ten novel peptides confirmed novel isoforms in five genes. Additionally, we found novel isoforms of IMPDH1, an IRD-associated gene, with supporting peptide evidence. This study provides a comprehensive overview of the transcript and protein isoforms expressed in the healthy human neural retina. Moreover, it highlights the importance of studying tissue specific transcriptomes in greater detail to better understand tissue-specific regulation and to identify disease-causing variants.
2024-10-17 | PXD045187 | Pride
Project description:We report a method for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using non-specific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing using PacBio of these chromatin stencils enables nucleotide-resolution readout of the primary architecture of multi-kilobase chromatin fibers (Fiber-seq).
2020-06-25 | GSE146940 | GEO