Ribo-Pop: simple, cost-effective, and widely applicable ribosomal RNA depletion

Thompson, Mary Kay; Kiourlappou, Maria; Davis, Ilan

doi:10.1261/rna.076562.120

Cited by 11 publications

(8 citation statements)

References 58 publications

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“…Systematic RNA-seq data comparison indicated that RNA-seq from rProbe libraries yielded a highly correlative transcriptomic landscape with that of the commonly used Illumina Ribo-Zero Kit and poly(A) selection method. However, about 3% of genes showed significant expression changes between rProbe and poly(A) selection method, as same as the disturbance have been reported (Morlan et al 2012 ; Zhao et al 2018 ; Thompson et al 2020 ). Culture condition and library preparation that makes the RNA pool differ in concentration and composition might be one of the explanations.…”

Section: Discussionsupporting

confidence: 80%

“…Thus, numerous rRNA-depletion methods have been developed including commercial kits such as Ribo-Zero Gold rRNA Removal Kit (Yeast), which has not yet launched new alternatives for fungi after discontinued. Several studies have reported the species-specific RNA-seq library construction methods that use hybrid-subtraction strategies for rRNA removal in higher eukaryotes and bacteria (Giannoukos et al 2012 ; Thompson et al 2020 ; Zeng et al 2020 ). Recently, a similar method has also been reported in Cryptococcus neoformans (Telzrow et al 2021 ).…”

Section: Discussionmentioning

confidence: 99%

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Efficient and specific DNA oligonucleotide rRNA probe-based rRNA removal in Talaromyces marneffei

Zhang

et al. 2022

Mycology

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Emerging evidence showed that lncRNAs play important roles in a wide range of biological processes of fungi such as Saccharomyces cerevisiae . However, systemic identification of lncRNAs in non-model fungi is a challenging task as the efficiency of rRNA removal has been proved to be affected by mismatches of universal rRNA-targeting probes of commercial kits, which forces deeper sequencing depth and increases costs. Here, we developed a low-cost and simple rRNA depletion method (rProbe) that could efficiently remove more than 99% rRNA in both yeast and mycelium samples of Talaromyces marneffei . The efficiency and robustness of rProbe were demonstrated to outperform the Illumina Ribo-Zero kit. Using rProbe RNA-seq, we identified 115 differentially expressed lncRNAs and constructed lncRNA-mRNA co-expression network related to dimorphic switch of T. marneffei. Our rRNA removal method has the potential to be a useful tool to explore non-coding transcriptomes of non-model fungi by adjusting rRNA probe sequences species specifically.

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Efficient and specific DNA oligonucleotide rRNA probe-based rRNA removal in Talaromyces marneffei

Zhang

et al. 2022

Mycology

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“…In the latter approach, DNA oligos complementary to rRNA facilitate their removal prior to library construction. These oligos can be biotinylated for magnetic bead affinity purification ( 6 , 22 , 23 ), commercialized for some taxa in the Ribo-Minus and the now discontinued Ribo-Zero Gold kits.…”

Section: Introductionmentioning

confidence: 99%

Optimized design of antisense oligomers for targeted rRNA depletion

Phelps

Carlson

Lee

2020

Nucleic Acids Research

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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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“…Samples were then pooled and purified over a Zymo Oligo Clean & Concentrator column (Genesee Scientific #11-380) according to manufacturer’s instructions. rRNA was subsequently depleted as previously described ( Thompson et al, 2020 ) using biotinylated oligos oSB6755-oSB6769. rRNA-depleted samples were then reverse transcribed in 20 uL reactions with primer NI802 and Protoscript II (200 U/μl) for 30 min at 50°C.…”

Section: Methodsmentioning

confidence: 99%

Microtubule integrity regulates budding yeast RAM pathway gene expression

Lee

Biggins

2022

Front. Cell Dev. Biol.

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During mitosis, cells must spatiotemporally regulate gene expression programs to ensure accurate cellular division. Failures to properly regulate mitotic progression result in aneuploidy, a hallmark of cancer. Entry and exit from mitosis is largely controlled by waves of cyclin-dependent kinase (CDK) activity coupled to targeted protein degradation. The correct timing of CDK-based mitotic regulation is coordinated with the structure and function of microtubules. To determine whether mitotic gene expression is also regulated by the integrity of microtubules, we performed ribosome profiling and mRNA-sequencing in the presence and absence of microtubules in the budding yeast Saccharomyces cerevisiae. We discovered a coordinated translational and transcriptional repression of genes involved in cell wall biology processes when microtubules are disrupted. The genes targeted for repression in the absence of microtubules are enriched for downstream targets of a feed-forward pathway that controls cytokinesis and septum degradation and is regulated by the Cbk1 kinase, the Regulation of Ace2 Morphogenesis (RAM) pathway. We demonstrate that microtubule disruption leads to aberrant subcellular localization of Cbk1 in a manner that partially depends on the spindle position checkpoint. Furthermore, constitutive activation of the RAM pathway in the absence of microtubules leads to growth defects. Taken together, these results uncover a previously unknown link between microtubule function and the proper execution of mitotic gene expression programs to ensure that cell division does not occur prematurely.

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Ribo-Pop: simple, cost-effective, and widely applicable ribosomal RNA depletion

Cited by 11 publications

References 58 publications

Efficient and specific DNA oligonucleotide rRNA probe-based rRNA removal in Talaromyces marneffei

Efficient and specific DNA oligonucleotide rRNA probe-based rRNA removal in Talaromyces marneffei

Optimized design of antisense oligomers for targeted rRNA depletion

Microtubule integrity regulates budding yeast RAM pathway gene expression

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