!6S rRNA targeted metagenome
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ABSTRACT:
PROVIDER: PRJEB25801 | ENA |
REPOSITORIES: ENA
Similar Datasets
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
Project description:Optimized design of antisense oligomers for targeted rRNA depletion
| PRJNA640808 | ENA
Project description:We used Targeted RNase H-mediated Extraction of crosslinked RBPs (TREX)to assess the endogenous region-specific binding partners of 45S rRNA in human HCT116 cells. We performed TREX experiments against the full-length 45S, as well as each individual region (5'ETS. 18S, ITS1, 5.8S, ITS2, 28S, and 3'ETS). Extracted proteins from RNase H digested and control cells (4 or 5 replicate per region per condition) were compared, using label-free (LFQ) Quantitative proteomics.
2023-12-27 | PXD044659 | Pride
Project description:Geisenheim Vineyard FACE rRNA Metagenome Metagenome
| PRJNA680929 | ENA
Project description:We tested a number of rRNA removal methods (Illumina RiboZero Plus, NEBNext, NEB Core Depletion Set with custom probes, siTools Panarchaea, siTools RiboPool) on 4 model halophile species: Halobacterium salinarum, Haloferax volcanii, Haloferax meditteranei, Haloarcula hispanica). It was found that methods using custom probes (NEB Core Depletion set with HVO probes, siTools RiboPool with HVO probes) efficiently remove rRNA in species they are targeted to, and that Panarchaea efficiently removes rRNA in all 4 tested species.
2022-04-14 | GSE200776 | GEO
Project description:mRNA sequencing in bacteria is challenging due to the abundance of ribosomal rRNA that cannot be easily removed prior to sequencing. While commercially available kits target specific rRNA sequences found in defined lists of common bacterial species, they are frequently inefficient when applied to other divergent species, including those from environmental isolates. Similar to the commercial kits, other common techniques for rRNA depletion typically employ large probe sets that tile full-length rRNA sequences; however, such approaches are both time consuming and expensive when applied to multiple species or complex consortia of non-model microbes. To overcome these limitations, we present EMBR-seq+, which employs less than twenty target oligonucleotides per rRNA molecule, and builds upon our previous rRNA depletion approach, EMBR-seq, through the addition of an RNase H depletion step, to achieve rRNA removal efficiencies of up to 99%. First, we applied EMBR-seq+ to monocultures of Escherichia coli, Geobacter metallireducens, and Fibrobacter succinogenes strain UWB7 to deplete rRNA to approximately 1-7% of the sequencing reads, demonstrating that the new method can be easily extended to diverse bacterial species. Further, in more complex co-cultures between F. succinogenes strain UWB7 and anerobic fungal species, we applied EMBR-seq+ to deplete both bacterial and fungal rRNA, with an approximately 4-fold improved bacterial rRNA depletion efficiency compared to a previous report using a commercial kit, thereby showing that the method can be effectively translated to non-model microbial mixtures. Notably, we also demonstrate that for microbial species with poorly annotated genomes and unknown rRNA sequences, the RNase H depletion component of EMBR-seq+ enables rapid iterations in probe design without requiring to start experiments from total RNA each time, and was key for depleting fungal rRNA to enrich the bacterial mRNA readout in co-cultures. Finally, efficient depletion of rRNA enabled systematic quantification of the reprogramming of the bacterial transcriptome when cultured in the presence of the anerobic fungi, Anaeromyces robustus and Caecomyces churrovis. We observed that F. succinogenes strain UWB7 transcribes nearly 200 carbohydrate-active enzyme (CAZyme) genes in both monoculture and co-culture conditions, with several lignocellulose-degrading CAZymes downregulated in the presence of an anerobic gut fungus. This finding is consistent with the premise that bacteria and fungi specialize in different aspects of biomass breakdown, such that the presence of one regulates the CAZyme production of the other. This also supports previous findings that the fungi release excess reducing sugars in the supernatant, which benefits other members of the microbial community. Thus EMBR-seq+ provides a new and detailed perspective of a rumen microbiome model system by dramatically improving the efficiency of mRNA sequencing, and more generally also enables high-throughput, cost-effective and rapid quantification of the transcriptome to gain functional insights into less-studied and non-model microbial systems.
2023-09-29 | GSE223404 | GEO
Project description:Cells expressing Tap-Tagged PUF3 were used for selection on IgG beads, then released using TEV protease. Samples were input and bound fraction without rRNA removal, and unbound fraction and input after rRNA removal with oligonucleotides and RNase H.
2019-06-28 | E-MTAB-8068 | biostudies-arrayexpress