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Optimized design of antisense oligomers for targeted rRNA depletion

ABSTRACT: Optimized design of antisense oligomers for targeted rRNA depletion

PROVIDER: PRJNA640808 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12060513_1.fastq.gz	Fastqsanger.gz
	SRR12060513_2.fastq.gz	Fastqsanger.gz
	SRR12060514_1.fastq.gz	Fastqsanger.gz
	SRR12060514_2.fastq.gz	Fastqsanger.gz
	SRR12060515_1.fastq.gz	Fastqsanger.gz

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Optimized design of antisense oligomers for targeted rRNA depletion

Project description:RNA sequencing (RNA-seq) has become a standard method for quantifying gene expression transcriptome-wide. Due to the extremely high proportion of ribosomal RNA (rRNA) in total RNA, sequencing libraries usually incorporate messenger RNA (mRNA) enrichment. Although polyadenylate (poly(A)) tail selection is widely used, many applications require alternate approaches such as rRNA depletion. Recently, selective rRNA digestion, using RNaseH and antisense DNA oligomers that tile the length of target RNAs, has emerged as an easy, cost-effective alternative to commercial rRNA depletion kits. Here, we present a streamlined RNaseH-mediated rRNA depletion method that uses shorter antisense oligos that only sparsely tile the target RNA, in a digestion reaction of only 5 minutes. We wrote a Web tool, Oligo-ASST, that simplifies oligo design to favor target regions with optimal thermodynamic properties, and additionally allows users to design common oligo pools that can simultaneously target divergent RNAs in their regions of higher sequence similarity. We demonstrate the efficacy of these oligos by building rRNA-depleted sequencing libraries for Xenopus laevis as well as zebrafish, which expresses two distinct versions of the 28S, 18S, 5.8S, and 5S rRNAs during embryogenesis. These libraries efficiently deplete rRNA to <5% of total reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available at https://mtleelab.pitt.edu/oligo to design antisense oligos for any taxon or to target any abundant RNA for depletion.

2020-06-28 | GSE152902 | GEO

Optimized design of antisense oligomers for targeted rRNA depletion.

Project description:RNA sequencing (RNA-seq) is extensively used to quantify gene expression transcriptome-wide. Although often paired with polyadenylate (poly(A)) selection to enrich for messenger RNA (mRNA), many applications require alternate approaches to counteract the high proportion of ribosomal RNA (rRNA) in total RNA. Recently, digestion using RNaseH and antisense DNA oligomers tiling target rRNAs has emerged as an alternative to commercial rRNA depletion kits. Here, we present a streamlined, more economical RNaseH-mediated rRNA depletion with substantially lower up-front costs, using shorter antisense oligos only sparsely tiled along the target RNA in a 5-min digestion reaction. We introduce a novel Web tool, Oligo-ASST, that simplifies oligo design to target regions with optimal thermodynamic properties, and additionally can generate compact, common oligo pools that simultaneously target divergent RNAs, e.g. across different species. We demonstrate the efficacy of these strategies by generating rRNA-depletion oligos for Xenopus laevis and for zebrafish, which expresses two distinct versions of rRNAs during embryogenesis. The resulting RNA-seq libraries reduce rRNA to <5% of aligned reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available at https://mtleelab.pitt.edu/oligo to design antisense oligos for any taxon or to target any abundant RNA for depletion.

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Predicting off-target effects of antisense oligomers targeting bacterial mRNAs with the MASON webserver

Project description:Antisense oligomers (ASOs) such as peptide nucleic acids (PNAs), designed to inhibit the translation of essential bacterial genes, have emerged as attractive sequence- and species-specific programmable RNA antibiotics. Yet, potential drawbacks include unwanted side effects caused by their binding to transcripts other than the intended target. To facilitate the design of PNAs with minimal off-target effects, we developed MASON (Make AntiSense Oligomers Now), a webserver for the design of PNAs that target bacterial mRNAs. MASON generates PNA sequences complementary to the translational start site of a bacterial gene of interest and reports critical sequence attributes and potential off-target sites. We based MASON’s off-target predictions on experiments in which we treated Salmonella enterica serovar Typhimurium with a series of 10mer PNAs derived from a PNA targeting the essential gene acpP but carrying two serial mismatches. Growth inhibition and RNA-sequencing (RNA-seq) data revealed that PNAs with terminal mismatches are still able to target acpP, suggesting wider off-target effects than anticipated. Comparison of these results to an RNA-seq dataset from uropathogenic Escherichia coli (UPEC) treated with eleven different PNAs confirmed our findings are not unique to Salmonella. We believe that MASON’s off-target assessment will improve the design of specific PNAs and other ASOs.

2022-07-01 | GSE199542 | GEO

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Project description:To validate the 244K 60-mer probes using a custom-designed Agilent oligo array and assuming antisense orientation of all target sequences. A total of four test arrays were designed assuming both sense and antisense orientations. A sole pooled RNA was used for testing all 4 designs. Two arrays were hybridized for each design. This series contains test results for design 2520023.

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2010-05-18 | E-GEOD-16550 | biostudies-arrayexpress

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2023-10-24 | PXD039394 | Pride

A comparison of the effects of duplex-specific nuclease, antisense oligonucleotides, and riboZero rRNA depletion on ribo-Seq data from mouse

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).

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Raw OBE MS data for Stepwise design of pseudosymmetric protein hetero-oligomers

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