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ABSTRACT:
PROVIDER: PRJNA598159 | ENA |
REPOSITORIES: ENA
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Project description:The measurement of RNA abundance derived from massively parallel sequencing experiments is an essential technique. Methods that reduce ribosomal RNA levels are usually required prior to sequencing library construction because ribosomal RNA typically comprises >90% of the total RNA molecules in a sample. For some experiments, ribosomal RNA depletion is favored over poly(A) selection because it offers a more inclusive representation of the transcriptome. However, methods to deplete ribosomal RNA are generally proprietary, complex, inefficient, applicable to only specific species, or compatible with only a narrow range of RNA input levels. Here, we describe Ribo-Pop (ribosomal RNA depletion for popular use), a simple workflow and antisense oligo design strategy that we demonstrate works over a wide input range and can be easily adapted to any organism with a sequenced genome. We provide a computational pipeline for probe selection, a streamlined 20-minute protocol, and ready-to-use oligo sequences for several organisms. We anticipate that our simple and generalizable “open source” design strategy would enable virtually any lab to pursue full transcriptome sequencing in their organism of interest with minimal time and resources.
2020-08-12 | GSE150332 | GEO
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:Next generation sequencing analysis was performed to identify potential target genes of lncRNA BLACAT2. Purified total RNA (3 μg) was used to deplete ribosomal RNA with Ribo-Zero rRNA Removal Kits (MRZMB126,Epicentre). Poly(A)+ RNA was purified with oligo(dT) magnetic beads and fragmented into short sequences. The library was prepared with TruSeq Stranded mRNA LT Sample Prep Kit (Cat. No.15032612,Illumina) according to manufacturer’s instructions. Each library was sequenced on an Illumina Hiseq2500 in 125PE mode (Illumina, San Diego, CA, USA). These data provided transcriptional changes in bladder cancer cell line UM-UC-3 after BLACAT2 depletion.
2018-02-01 | GSE101287 | GEO
Project description:Human ribosomes are made of around 80 ribosomal proteins (RPs) and four ribosomal RNAs. To explore gene expression regulation by RPs at the transcriptional and translational levels, parallel RNA-seq and Ribo-seq were conducted in A549 cells after knockdown of individual RP.
2022-01-27 | GSE168445 | GEO
Project description:Ribo-Pop: Simple, cost-effective, and widely applicable ribosomal RNA depletion
| PRJNA631775 | ENA
Project description:A major challenge for RNA-seq analysis of gene expression is to achieve sufficient coverage of informative non-ribosomal transcripts. In eukaryotic samples, this is typically achieved by selective oligo(dT)-priming of messenger RNAs to exclude ribosomal RNA (rRNA) during cDNA synthesis. However, this strategy is not compatible with prokaryotes in which functional transcripts are generally not polyadenylated. To overcome this, we adopted DASH (Depletion of Abundant Sequences by Hybridization), initially developed for eukaryotic cells, to improve both the sensitivity and depth of bacterial RNA-seq. DASH employs the Cas9 nuclease to remove unwanted cDNA sequences prior to library amplification. We report the design, evaluation, and optimization of DASH experiments for standard bacterial short-read sequencing approaches, including software for automated guide RNA (gRNA) design for Cas9-mediated cleavage in bacterial rDNA sequences. Using these gRNA pools, we effectively removed rRNA reads (56-86%) in RNA-seq libraries from two different model bacteria, the Gram-negative pathogen Salmonella enterica and the anaerobic gut commensal Bacteroides thetaiotaomicron. DASH works robustly, even with sub-nanogram amounts of input cDNA. Its efficiency, high sensitivity, ease of implementation, and low cost (~$5 per sample) render DASH an attractive alternative to rRNA removal protocols, in particular for material-constrained studies where conventional ribodepletion techniques fail.
2020-05-11 | GSE147155 | GEO
Project description:Purpose: We wanted to determine the transcriptome profile of PAO1 wild type, PAO1ΔrhlE1, PAO1ΔrhlE2 and PAO1ΔrhlE1ΔrhlE2 mutants on swarming conditions. Method: Cells were harvested using the RNA bacteria protect solution (QIAGEN). Total RNAs was extracted and purified using Monarch RNA isolation kit (NEB), treated with DNase I (Promega) three times to remove contaminating genomic DNA and re-purified again using phenol-chloroform. The removal of ribosomal RNA was performed using the Ribo-Zero Bacteria Kit (Illumina) and cDNA libraries were generated with the ScriptSeq v2 Kit (Illumina) . The samples were sequenced in single end mode on an Illumina HiSeq 4000 device and mRNA reads were trimmed and mapped to the NC_002516.2(PAO1) reference genome from NCBI using Stampy pipeline with defaut settings.
2021-02-18 | GSE166986 | GEO
Project description:In this study, we systematically identified RNAs associated with ribosomes. To identify ribosome associated RNAs, C-terminal ZZ-tagged Rpl16a or Rpl16b, expressed under control of thier native promoter, were affinity purified from whole cell extracts of cultures grown to mid-log phase in minimal medium. Extracts were incubated with immunoglobulin G (IgG) coupled microbeads, washed, and ribosomes were eluted by tobacco etch virus (TEV) protease treatment. We performed two biological replicates with each protein and analyzed the RNA content using oligo microarrays. Total RNA isolated from extracts of cells expressing Rpl16a-ZZ or Rpl16b-ZZ (input) and from the affinity-purified ribosomes was reverse transcribed with oligo(dT) primers. cDNA was labeled with Cy3 and Cy5 fluorescent dyes, respectively, and competitively hybridized to yeast oligo microarrays. Alternatively, RNA was isolated from sucrose gradient fractions containing 60S subunits, 80S monosomes and polysomes. Here, we performed four biological replicates. Analysis was done with oligo microarrays as described above. A strain or line experiment design type assays differences between multiple strains, cultivars, serovars, isolates, lines from organisms of a single species.
2009-05-12 | GSE13654 | GEO
Project description:We report two knockouts of the CASC3 (Barentsz, MLN51) gene and further depletion of gene expression with small interfering RNAs (siRNAs). CASC3 is a component of the exon-junction complex (EJC) which is deposited upstream of splice junctions on the mRNA. The EJC core and peripheral interacting factors are involved in RNA splicing, export, translation and nonsense-mediated decay. Contrasting to the other factors that form the exon-junction complex, CASC3 individual role on these processes was relatively poorly understood in the literature. To further elucidate the role of CASC3 in these processes we have established 2 different CRISPR-Cas9 genetic knockouts (KO) of CASC3. We extracted the total RNA by using the NucleoSpin RNA Plus kit (Macherey-Nagel), and performed ribosomal depletion and strand-specific library preparation with the TruSeq Stranded Total RNA protocol with Ribo-Zero Gold. Sequencing of the KO clones as well as the KO clones treated with CASC3 siRNA was carried out with the Illumina NovaSeq6000 sequencer with 2×100bp, targeting approximately 50 million read-pairs per sample. By studying the RNA-Seq of the 4 conditions, one with minimal CASC3 expression (condition H) or and three with complete depletion of the protein expression (conditions H-KD, T, T-KD), we observed the up-regulation of known and novel nonsense mediated decay substrates, thus establishing CASC3 as a critical factor for efficient nonsense mediated decay of certain transcripts.
2020-07-03 | E-MTAB-8461 | biostudies-arrayexpress
Project description:Stranded RNA-seq were performed on total RNA following ribosomal RNAs depletion (Ribo-zero removal kit, illumina) for glioblastoma stem cell
| PRJNA678319 | ENA