Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1138001 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29898424_1.fastq.gz | Fastqsanger.gz | |||
| SRR29898425_1.fastq.gz | Fastqsanger.gz | |||
| SRR29898426_1.fastq.gz | Fastqsanger.gz | |||
| SRR29898427_1.fastq.gz | Fastqsanger.gz | |||
| SRR29898428_1.fastq.gz | Fastqsanger.gz |
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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:Adenovirus is a common human pathogen that relies on host cell processes for transcription and processing of viral RNA and protein production. Although adenoviral promoters, splice junctions, and cleavage and polyadenylation sites have been characterized using low-throughput biochemical techniques or short read cDNA-based sequencing, these technologies do not fully capture the complexity of the adenoviral transcriptome. By combining Illumina short-read and nanopore long-read direct RNA sequencing approaches, we mapped transcription start sites and cleavage and polyadenylation sites across the adenovirus genome. In addition to confirming the known canonical viral early and late RNA cassettes, our analysis of splice junctions within long RNA reads revealed an additional 35 novel viral transcripts. These RNAs include fourteen new splice junctions which lead to expression of canonical open reading frames (ORF), six novel ORF-containing transcripts, and fifteen transcripts encoding for messages that potentially alter protein functions through truncations or fusion of canonical ORFs. In addition, we also detect RNAs that bypass canonical cleavage sites and generate potential chimeric proteins by linking separate gene transcription units. Of these, an evolutionary conserved protein was detected containing the N-terminus of E4orf6 fused to the downstream DBP/E2A ORF. Loss of this novel protein, E4orf6/DBP, was associated with aberrant viral replication center morphology and poor viral spread. Our work highlights how long-read sequencing technologies can reveal further complexity within viral transcriptomes.
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Project description:The Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo pathway and essential mechanotransducer. It has been suggested to play a crucial role for early embryo development, but the relevance for early germ layer commitment of human induced pluripotent stem cells (iPSCs) remains largely unclear. To gain better insight into the functional relevance of YAP1 in these early cell-fate decisions, we generated iPSC lines with YAP1 knockout (YAP-/-) with CRISPR/Cas9 technology and analyzed transcriptomic and epigenetic modifications. In YAP-/- iPSCs the expression of several YAP1 targets changed and NODAL, which is an important regulator of cell differentiation, was upregulated. Furthermore, YAP1 deficiency evoked global DNA methylation changes. Directed differentiation of adherent iPSC colonies toward endoderm, mesoderm, and ectoderm could be induced, albeit endodermal and ectodermal differentiation showed transcriptomic and epigenetic changes in YAP-/- lines. Notably, in self-organized embryoid bodies (EBs) germ layer specification was clearly impaired. This phenotype was rescued via lentiviral overexpression of YAP1 and in tendency also by NODAL inhibitors. Our results demonstrate that YAP1 plays an important role during early germ layer specification of iPSCs, particularly for the non-directed self-organization of EBs, and this is at least partly attributed to activation of the NODAL pathway.
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Project description:The Yes-associated protein 1 (YAP1) is a downstream effector of the Hippo pathway and essential mechanotransducer. It has been suggested to play a crucial role for early embryo development, but the relevance for early germ layer commitment of human induced pluripotent stem cells (iPSCs) remains largely unclear. To gain better insight into the functional relevance of YAP1 in these early cell-fate decisions, we generated iPSC lines with YAP1 knockout (YAP-/-) with CRISPR/Cas9 technology and analyzed transcriptomic and epigenetic modifications. In YAP-/- iPSCs the expression of several YAP1 targets changed and NODAL, which is an important regulator of cell differentiation, was upregulated. Furthermore, YAP1 deficiency evoked global DNA methylation changes. Directed differentiation of adherent iPSC colonies toward endoderm, mesoderm, and ectoderm could be induced, albeit endodermal and ectodermal differentiation showed transcriptomic and epigenetic changes in YAP-/- lines. Notably, in self-organized embryoid bodies (EBs) germ layer specification was clearly impaired. This phenotype was rescued via lentiviral overexpression of YAP1 and in tendency also by NODAL inhibitors. Our results demonstrate that YAP1 plays an important role during early germ layer specification of iPSCs, particularly for the non-directed self-organization of EBs, and this is at least partly attributed to activation of the NODAL pathway.
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