Ontology highlight
ABSTRACT:
PROVIDER: PRJNA883055 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR21672341.fastq.gz | Fastqsanger.gz | |||
| SRR21672342.fastq.gz | Fastqsanger.gz | |||
| SRR21672343.fastq.gz | Fastqsanger.gz | |||
| SRR21672344.fastq.gz | Fastqsanger.gz | |||
| SRR21672345.fastq.gz | Fastqsanger.gz |
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Project description:Ribosome profiling has become an essential tool for studying mRNA translation in cells with codon-level resolution. However, its widespread application remains hindered by the labour-intensive workflow, low efficiency and high costs associated with sequencing sample preparation. Here, we present a new cost-effective and ultra-sensitive library preparation method that significantly advances the applicability of ribosome profiling. By implementing bead-coupled enzymatic reactions and product purifications, our approach increases both yield and throughput while maintaining high reproducibility. Demonstrating the sensitivity of the protocol we prepared libraries from as little as 12 fmol of RNA, which expands the feasibility of ribosome profiling from minimal input samples, such as derived from small populations, stressed cells, or patient-derived specimens. Additionally, we validate the versatility of the protocol across multiple species and demonstrate its applicability for RNA-seq library preparation. Altogether, this protocol provides a highly accessible and efficient alternative to existing ribosome profiling workflows, facilitating research in previously challenging experimental contexts.
2025-08-19 | GSE294467 | GEO
Project description:Ribo-ITP coupled ribosome profiling identifies non-canonical translational events in low input samples
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Project description:In the last decade, an unexpectedly large number of translated regions (translons) have been discovered using ribosome profiling and proteomics. Translons can regulate mRNA translation and encode micropeptides that contribute to multiprotein complex formation, Ca2+ regulation in muscle, and signaling during embryonic development. However, identification of translons has been limited to cell lines or large organs due to high input requirements for conventional ribosome profiling and mass spectrometry. Here, we employed Ribo-ITP, a low input ribosome profiling technique on difficult to collect samples like microdissected hippocampal tissues and single pre-implantation embryos. We detected thousands of translons across these samples, most of which initiated at non-canonical start sites, were less than 100 amino long with significantly altered amino acid composition. Comparative analysis of more than a thousand ribosome profiling datasets across a wide range of cell types revealed pervasive expression of most of the detected translons. To test the translational capacity of the detected translons, we engineered a translon dependent GFP reporter system, and detected robust expression of translons initiating at near-cognate start codons in mouse embryonic stem cells (mESCs). Mutating the translons in mESCs identified a small proportion that negatively impacted growth. Taken together, we present a proof-of-concept study to identify non-canonical translation events from ultra-low input samples which can be applied to cell and tissue types inaccessible to conventional methods.
2025-08-05 | GSE300869 | GEO
Project description:In the last decade, an unexpectedly large number of translated regions (translons) have been discovered using ribosome profiling and proteomics. Translons can regulate mRNA translation and encode micropeptides that contribute to multiprotein complex formation, Ca2+ regulation in muscle, and signaling during embryonic development. However, identification of translons has been limited to cell lines or large organs due to high input requirements for conventional ribosome profiling and mass spectrometry. Here, we employed Ribo-ITP, a low input ribosome profiling technique on difficult to collect samples like microdissected hippocampal tissues and single pre-implantation embryos. We detected thousands of translons across these samples, most of which initiated at non-canonical start sites, were less than 100 amino long with significantly altered amino acid composition. Comparative analysis of more than a thousand ribosome profiling datasets across a wide range of cell types revealed pervasive expression of most of the detected translons. To test the translational capacity of the detected translons, we engineered a translon dependent GFP reporter system, and detected robust expression of translons initiating at near-cognate start codons in mouse embryonic stem cells (mESCs). Mutating the translons in mESCs identified a small proportion that negatively impacted growth. Taken together, we present a proof-of-concept study to identify non-canonical translation events from ultra-low input samples which can be applied to cell and tissue types inaccessible to conventional methods.
2025-08-05 | GSE300832 | GEO
Project description:Dysregulated protein synthesis is a major underlying cause of many neurodevelopmental diseases such as Fragile X Syndrome. A very robust technique is required to capture subtle but biologically significant differences in neurological disorders. Ribosome profiling, which is based on deep sequencing of mRNA fragments protected from ribonuclease digestion by ribosomes, is a powerful tool to study translational control. However, it has been mainly applied to rapidly dividing cells where translation is robust and where large amounts of starting material are readily available. The application of ribosome profiling to low-input brain tissue where translation is modest and where gene expression changes between genotypes are expected to be small has not been carefully evaluated. Using hippocampal tissue from wide type and fragile X mental retardation 1 (Fmr1) knockout mice, we show that variable RNase digestion can lead to significant sample batch effects. We also establish GC content and ribosome footprint length as quality control metrics for ribonuclease digestion. We performed ribonuclease titration experiments for low-input samples to identify optimal conditions for this critical step that is often improperly conducted. Our data reveal that optimal RNase digestion is essential to ensure high quality and reproducibility of ribosome profiling especially for low-input brain tissue.
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Project description:Ribo-ITP coupled ribosome profiling identifies non-canonical translational events in low input samples [Ribo-seq]
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Project description:Despite advancement of data-independent acquisition mass spectrometry (DIA-MS) to provide comprehensive and reproducible quantitative proteome profiling, its utility in very low-input samples is limited. For low-input samples at microscale, the proteome composition and overall peptide ion patterns are different from the conventional sample size. Thus, the conventional LC-MS/MS acquisition and widely used large proteome-scale DIA libraries may not be suitable for the microscale sample. In this study, we report a sample size-comparable library-based DIA approach for enhanced proteome coverage of low-input samples at nanoscale to near single-cell levels (i.e. nanogram cells,~5-50 cells). With these spectral libraries made freely available, we envision that such single-shot DIA strategy and the DIA digital map will advance quantitative proteomics applications by enabling improved deep proteome profiling for mass-limited samples.
2022-11-07 | PXD027679 | JPOST Repository
Project description:We developed a high-throughput sequencing protocol that efficiently captures small RNAs from low input samples while minimizing inherent biases associated with library production. The protocol was builds on previously published method that adds a barcode to samples at the earliest step in preparation such that all downstream manipulations can be performed on a pool of many samples thereby greatly reducing reagent usage and workload. We optimized adapter concentrations and showed modified adapters greatly increases the efficiency of small RNA capture. This work also demonstrates that unique molecular identifiers incorporated into the adapters can be used to bioinformatically remove PCR duplication.
2019-01-31 | GSE109162 | GEO
Project description:HFFF cells were either mock or WT strain 17 HSV-1 infected for 8 h. Samples were processed according to the original ChIPmentation protocol (Schmidl C et al. ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors. Nat Methods. 2015) with few changes as explained in the Materials and Methods section.
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