Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA279866 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Thermostable Group II intron reverse transcriptase single-stranded DNA-seq
| PRJNA386569 | ENA
Project description:RNA-Seq of human reference RNA samples using a thermostable group II intron reverse transcriptase
| PRJNA301711 | ENA
Project description:Inflammatory breast cancer (IBC), the most aggressive and lethal breast cancer subtype, lacks unequivocal genomic differences or robust biomarkers that differentiate it from non-IBC. Here, Thermostable Group II Intron Reverse Transcriptase sequencing of RNA samples revealed myriad differences in tumors, PBMCs, and plasma that distinguished IBC from non-IBC patients and healthy donors across all tested non-IBC subtypes. By mapping reads to genome and transcriptome reference sequences and quantitating intron-to-exon read depth ratios, we developed methods for parallel analysis of transcriptional and post-transcriptional gene regulation. This analysis identified numerous protein-coding genes in IBC patient tumors and PBMCs with high intron-to-exon read depths, suggesting rate-limiting RNA splicing that negatively impacts mRNA production. Mirroring gene expression differences in tumors and PBMCs, over-represented protein-coding gene RNAs in IBC patient plasma were largely intron RNA fragments, while those in non-IBC patient and healthy donor plasma were largely mRNA fragments. Our findings provide new insights into IBC and should enable monitoring disease progression by liquid biopsy.
2026-05-01 | GSE325047 | GEO
Project description:Here we present dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq), which encodes DMS modifications as mismatches using a thermostable group II intron reverse transcriptase (TGIRT). DMS-MaPseq yields a high signal-to-noise ratio, can report multiple structural features for each molecule, and allows genome-wide studies as well as focused investigations of low abundance RNAs. We apply DMS-MaPseq to Drosophila melanogaster ovaries—the first experimental analysis of RNA structure in an animal tissue—and demonstrate its utility in the discovery of a functional RNA structure involved in the non-canonical GUG translation initiation of the human FXR2 mRNA. Additionally, we use DMS-MaPseq to compare the in vivo structure of messages in their pre-mRNA and mature forms. These applications illustrate DMS-MaPseq’s capacity to dramatically expand our ability to monitor RNA structure in vivo.
2016-09-16 | GSE84537 | GEO
Project description:The quality of RNA sequencing data relies on specific priming by the primer used for reverse transcription (RT-primer). Non-specific annealing of the RT-primer to the RNA template can generate reads with incorrect cDNA ends and can cause misinterpretation of data (RT mispriming). This kind of artifact in RNA-seq based technologies is underappreciated and currently no adequate tools exist to computationally remove them from published datasets. We show that mispriming can occur with as little as 2 bases of complementarity at the 3' end of the primer followed by intermittent regions of complementarity. We propose an experimental solution to avoid RT-mispriming by performing RNA-seq using thermostable group II intron derived reverse transcriptase (TGIRT-seq).
2018-06-26 | GSE85163 | GEO
Project description:5’ ends are important for determining the fate of RNA molecules. BCDIN3D is an RNA phospho-methyltransferase that methylates the 5’ monophosphate of specific RNAs. In order to gain new insights into the molecular function of BCDIN3D, we performed an unbiased analysis of its interacting RNAs by Thermostable Group II Intron Reverse Transcriptase coupled to next generation sequencing (TGIRT-seq). Our analyses showed that BCDIN3D interacts with full-length phospho-methylated tRNAHis and miR-4454. Interestingly, we found that miR-4455 is not synthesized from the annotated genomic locus, which is a primer-binding site for an endogenous retrovirus, but rather by Dicer cleavage of mature tRNAHis. Sequence analysis revealed that miR-4454 is identical to the 3’ end of tRNAHis. Moreover, we were able to generate this “miRNA” in vitro through incubation of mature tRNAHis with Dicer. As found previously for several pre-miRNAs, a 5’P-tRNAHis appears to be a better substrate for Dicer cleavage than a phospho-methylated tRNAHis. Moreover, tRNAHis 3’-fragment/”miR-4454” levels increase in cells depleted for BCDIN3D. Altogether, our results show that in addition to microRNAs, BCDIN3D regulates tRNAHis 3’-fragment processing without negatively affecting tRNAHis’s canonical function of aminoacylation.
2019-07-09 | GSE130951 | GEO
Project description:A wide range of sequencing methods have been developed to assess nascent RNA transcription and resolve the single-nucleotide position of RNA polymerase genome-wide. These techniques are often burdened with high input material requirements and lengthy protocols. We leveraged the template-switching properties of thermostable group II intron reverse transcriptase (TGIRT) and developed BuTT-Seq (BUlk analysis of nascent Transcript Termini sequencing), which can produce libraries from purified nascent RNA in 6 hours and from as few as 10,000 cells – an improvement of at least 10-fold over existing techniques. BuTT-Seq shows that inhibition of the superelongation complex (SEC) causes promoter-proximal pausing to move upstream in a fashion correlated with subnucleosomal fragments. To address transcriptional regulation in a tissue, BuTT-Seq was used to measure the circadian regulation of transcription from fly heads. All the results indicate that BuTT-Seq is a simple and powerful technique to analyze transcription at a high level of resolution.
2023-03-30 | GSE228595 | GEO
Project description:Thermostable reverse transcriptases are workhorse enzymes underlying nearly all modern techniques for RNA structure mapping and for transcriptome-wide discovery of RNA chemical modifications. Despite their wide use, these enzymes’ behaviors at chemical modified nucleotides remain poorly understood. Wellington-Oguri et al. recently reported an apparent loss of chemical modification within putatively unstructured polyadenosine stretches modified by dimethyl sulfate or 2’ hydroxyl acylation, as probed by reverse transcription. Here, re-analysis of these and other publicly available data, capillary electrophoresis experiments on chemically modified RNAs, and nuclear magnetic resonance spectroscopy on A 12 and variants show that this effect is unlikely to arise from an unusual structure of polyadenosine. Instead, tests of different reverse transcriptases on chemically modified RNAs and molecules synthesized with single 1-methyladenosines implicate a previously uncharacterized reverse transcriptase behavior: near-quantitative bypass through chemical modifications within polyadenosine stretches. All tested natural and engineered reverse transcriptases (MMLV; SuperScript II, III, and IV; TGIRT-III; and MarathonRT) exhibit this anomalous bypass behavior. Accurate DMS-guided structure modeling of the polyadenylated HIV-1 3´ untranslated region RNA requires taking into account this anomaly. Our results suggest that poly(rA-dT) hybrid duplexes can trigger unexpectedly effective reverse transcriptase bypass and that chemical modifications in poly(A) mRNA tails may be generally undercounted.
2020-04-22 | GSE149061 | GEO
Project description:Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq
| PRJNA575026 | ENA