Reverse transcription comparison
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB32237 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:We assess the mRNA-enrichment performance of a custom-made non-mRNA depletion protocol in comparison to a commercially available mRNA-enrichment kit (Ribo-off, Vazyme). Whereas most available kits focus only on removal of rRNA, our method also targets the transfer-messenger RNA (tmRNA). tmRNA was shown to consume up to 25% of the reads in RNA-sequencing data of Pseudomonas aeruginosa. Our established depletion technique is based on the targeting of overly abundant RNA species (16S and23S rRNA, tmRNA) in total RNA preparations of Pseudomonas aeruginosa PA14 by hybridization with organism-specific DNA probes and subsequent degradation by RNase H treatment. While introducing no systematic bias into the gene expression profile we were able to increase the mRNA read share of the total reads in the samples treated with our mRNA-enrichment protocol to 93% - 99%. Therefore, our custom-made depletion technique outcompeted the commercially available reference kit (72% mRNA share) and represents a cost-efficient mRNA-enrichment method for high-throughput next-generation sequencing.
2020-03-31 | GSE147748 | GEO
Project description:To investigate gene specificity at the level of translation in both the human genome and viruses, we devised a high-throughput bicistronic assay to quantify cap-independent translation. We uncovered thousands of novel cap-independent translation sequences, and we provide insights on the landscape of translational regulation in both humans and viruses. We find extensive translational elements in the 3′ untranslated region of human transcripts and the polyprotein region of uncapped RNA viruses. Through the characterization of regulatory elements underlying cap-independent translation activity, we identify potential mechanisms of secondary structure, short sequence motif,and base pairing with the 18S ribosomal RNA (rRNA). Furthermore, we systematically map the 18S rRNA regions for which reverse complementarity enhances translation. Thus, we make available insights into the mechanisms of translational control in humans and viruses.
2015-10-23 | GSE74277 | GEO
Project description:Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. A key step is the depletion of ribosomal RNA that allows sufficient depth of sequencing of the mRNA undergoing ribosomal translation. These data are designed to compare four strategies for ribosomal depletion; a novel strategy based on duplex-specific nuclease using either one or two cycles, the antisense oligonucleotides described in Ingola et al (2012) and the commercially available RiboZero kit (with a no treatment control).
2015-10-16 | E-MTAB-3583 | biostudies-arrayexpress
Project description:Crosslinking and immunoprecipitation (CLIP) followed by high-throughput sequencing identifies the binding sites of RNA binding proteins on RNAs. The covalent RNA-amino acid adducts produced by UV irradiation can cause premature reverse transcription termination, which may decrease overall cDNA yield but is exploited in state of the art CLIP methods to identify these crosslink sites at single-nucleotide resolution. Here, we show the ratio of read-through versus termination is highly dependent on the identity of the reverse transcriptase enzyme as well as on buffer conditions used. Commonly used enzymes, including Superscript III and AffinityScript, show high termination rates in standard magnesium buffer conditions, but show a single base difference in the position of termination for TARDBP motifs. In contrast, manganese-containing buffer promoted read-through at the adduct site. These results validate the use of standard enzymes and suggest that alternative enzyme and buffer choices for particularly challenging samples that contain extensive RNA adducts or other modifications that inhibit standard reverse transcription.
2017-08-10 | GSE101938 | GEO
Project description:N1-methyl adenosine (m1A) is a wide-spread RNA modification present in tRNA, rRNA and mRNA. m1A modification sites in tRNAs are evolutionary conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m1A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m1A can notably modulate RNA structure. It also blocks Watson-Crick base pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high throughput sequencing methods have been developed to sequence m1A. In this study, we introduce a reduction-based m1A sequencing (red-m1A-seq). We report that NaBH4 reduction of m1A can improve the mutation and readthrough rates using commercially available RT enzymes to give better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m1A to m6A to provide good controls, allowing the detection of m1A with higher sensitivity and accuracy. We applied red‑m1A-seq to sequence human small RNA and we not only detected all the previously reported tRNA m1A sites, but also new m1A sites in mt-tRNAAsn-ATT and 5.8S rRNA.
2024-04-24 | GSE253657 | GEO
Project description:Comparison of commercially available histone antibodies
| PRJNA278079 | ENA
Project description:Standard method of RNA-Seq captures mRNA by poly(A) capturing using Oligo dT beads, which is not suitable for degraded RNA. Here, we used three commercially available RNA-Seq library preparation kits (SMART-Seq, xGen Broad-range and RamDA-Seq) using random primer instead of Oligo dT beads. To evaluate the performance of these methods, we compared that the correla-tion, the number of detected expressing genes and the expression levels with Standard RNA-Seq method.SMART-Seq with rRNA depletion has relative advantages for RNA-Seq using low input and de-graded RNA.
2024-06-20 | GSE266382 | GEO
Project description:While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a high-throughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein-rRNA interactions, providing intriguing insights into the temporal order of protein-rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.Ribosome biogenesis is a dynamic process that involves the ordered assembly of ribosomal proteins and numerous RNA structural rearrangements. Here the authors apply ChemModSeq, a high-throughput RNA structure probing method, to quantitatively measure changes in RNA flexibility during the nucleolar stages of 60S assembly in yeast.
2017-10-04 | GSE83821 | GEO
Project description:RNA modifications are key regulatory factors for several biological and pathological processes. They are abundantly represented on ribosomal RNA (rRNA), where they contribute to regulate ribosomal function in mRNA translation. Altered RNA modification pathways have been linked to tumorigenesis as well as to other human diseases. In this study we quantitatively evaluated the site-specific pseudouridylation pattern in rRNA in breast cancer samples exploiting the RBS-Seq technique involving RNA bisulfite treatment coupled with a new NGS approach. We found a wide variability among patients at different sites. The most dysregulated positions in tumors turned out to be hypermodified with respect to a reference RNA. As for 2’O-methylation level of rRNA modification, we detected variable and stable pseudouridine sites, with the most stable sites being the most evolutionary conserved. We also observed that pseudouridylation levels at specific sites are related to some clinical and bio-pathological tumor features and are able to distinguish different patient clusters. This study is the first example of the contribution that newly available high-throughput approaches for site specific pseudouridine detection can provide to the understanding of the intrinsic ribosomal changes occurring in human tumors.
2023-06-07 | GSE220967 | GEO