Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Improved RNA preparation for RNA-seq of the intracellular bacterium Wolbachia wAlbB

ABSTRACT: Despite advances in RNA-seq, investigating the transcriptome of intracellular bacteria remains challenging due to the substantial presence of host RNA. In the case of Wolbachia spp. that are propagated in insect cell lines, commercially available rRNA depletion kits are often not suitable. Here, we describe a method to study the transcriptome of Wolbachia wAlbB in the Aedes albopictus cell line C6/36. To enrich the bacterial mRNA, eukaryotic mRNA was depleted by Dynabeads (Thermo Fisher Scientific). Then custom-designed riboPOOLs (siTOOLs Biotech) were used to remove both prokaryotic and eukaryotic rRNA. Compared to RNA prepared using the Illumina Ribo-Zero Plus rRNA Depletion Kit alone, additional depletion of eukaryotic mRNA increased wolbachial reads 7-fold to 0.7% of all reads. After removing eukaryotic and prokaryotic rRNAs with custom-designed riboPOOLs, there was a 300-fold increase of reads that mapped to Wolbachia (30.2%). Combining customized rRNA depletion from both organisms with eukaryotic mRNA depletion was more cost-effective than simply increasing the number of sequencing reads. This method can potentially be used for the enrichment of bacterial mRNA in studies of intracellular bacteria that cannot be propagated in standard cell lines.

ORGANISM(S): Aedes albopictus

PROVIDER: GSE297421 | GEO | 2026/02/11

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies

Project description:These experiments were designed to quantify depletion of rRNA sequencing reads from bacterial RNA-seq libraries and verify that mRNA sequencing reads were not altered. Specifically, we tested an rRNA depletion method using custom-designed biotinylated oligonucleotides and compared these results to undepleted (total RNA) libraries and libraries made with the previously-available Ribo-Zero kit (Illumina).

2019-12-28 | GSE142656 | GEO

Organism-specific depletion of highly abundant RNA species from bacterial total RNA

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

Mycobacterium tuberculosis H37Rv

Project description:Prokaryotic rRNA Depletion

| PRJNA182521 | ENA

Escherichia coli K-12

Project description:Prokaryotic rRNA Depletion

| PRJNA182522 | ENA

Comparative evaluation for the globin gene depletion methods for mRNA sequencing using the whole blood-derived total RNAs

Project description:Background: There are challenges in generating mRNA-Seq data from whole-blood derived RNA as globin gene and rRNA are frequent contaminants. Given the abundance of erythrocytes in whole blood, globin genes comprise some 80% or more of the total RNA. Therefore, depletion of globin gene RNA and rRNA are critical steps required to have adequate coverage of reads mapping to the reference transcripts and thus reduce the total cost of sequencing. In this study, we directly compared the performance of probe hybridization (GLOBINClear Kit and Globin-Zero Gold rRNA Removal Kit) and RNAse-H enzymatic depletion (NEBNext® Globin & rRNA Depletion Kit and Ribo-Zero Plus rRNA Depletion Kit) methods from 1 ug of whole blood-derived RNA on mRNA-Seq profiling. All RNA samples were treated DNaseI for additional cleanup before the depletion step and were processed for poly-A selection for library generation. Results: Probe hybridization revealed a better overall performance than the RNAse-H enzymatic depletion method, detecting a higher number of genes and transcripts without 3’ region bias. After depletion, samples treated with probe hybridization showed globin genes at 0.5% (±0.6%) of the total mapped reads; the RNAse-H enzymatic depletion had 3.2% (±3.8%). Probe hybridization showed more junction reads and transcripts compared with RNAse-H enzymatic depletion and also had a higher correlation (R>0.9) than RNAse-H enzymatic depletion (R>0.85). Conclusion: In this study, our results showed that 1 µg of high-quality RNA from whole blood could be routinely used for transcriptional profiling analysis studies with globin gene and rRNA depletion pre-processing. We also demonstrated that the probe hybridization depletion method is better suited to mRNA sequencing analysis with minimal effect on RNA quality during depletion procedures.

2020-11-30 | GSE150587 | GEO

Testing different rRNA removal methods in 4 species of model halophilic archaea

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

A cap 0-dependent mRNA capture method to analyze the yeast transcriptome

Project description:Analysis of the protein coding transcriptome by the RNA sequencing requires either enrichment of the desired fraction of coding transcripts or depletion of the abundant non-coding fraction consisting mainly of rRNA. We propose an alternative mRNA enrichment strategy based on the RNA-binding properties of the human IFIT1, an antiviral protein recognizing cap 0 RNA. Here, we compare for Saccharomyces cerevisiae an IFIT1-based mRNA pull-down with yeast targeted rRNA depletion by the RiboMinus method. IFIT1-based RNA capture depletes rRNA more effectively, producing high quality RNA-seq data with an excellent coverage of the protein coding transcriptome, while depleting cap-less transcripts such as mitochondrial or some non-coding RNAs. We propose IFIT1 as a cost effective and versatile tool to prepare mRNA libraries for a variety of organisms with cap 0 mRNA ends, including diverse plants, fungi and eukaryotic microbes.

2022-08-04 | GSE210198 | GEO

RNA-seq of iron-rich pelagic aggregates (iron snow) in 2017 and 2018

Project description:Iron-rich pelagic aggregates (iron snow) were collected directly onto silicate glass filters using an electronic water pump installed below the redoxcline. RNA was extracted and library preparation was done using the NEBNext Ultra II directional RNA library prep kit for Illumina. Data was demultiplied by GATC sequencing company and adaptor was trimmed by Trimgalore. After trimming, data was processed quality control by sickle and mRNA/rRNA sequences were sorted by SortmeRNA. mRNA sequences were blast against NCBI-non redundant protein database and the outputs were meganized in MEGAN to do functional analysis. rRNA sequences were further sorted against bacterial/archeal 16S rRNA, eukaryotic 18S rRNA and 10,000 rRNA sequences of bacterial 16S rRNA, eukaryotic 18S rRNA were subset to do taxonomy analysis.

2021-10-15 | E-MTAB-9686 | biostudies-arrayexpress

Quantitative mRNA profile of eif3e (yin6, int6) deleted S. pombe cells

Project description:Int6/yin6 encodes the eIF3e subunits of the eukaryotic translation initiation factor 3 complex (eIF3). To assess the impact of eIF3e on the global transcriptome, RNA seq of total mRNA was performed. Total RNA was prepared from wildtype S. pombe (h- ura4-D18 leu1-32) and cells deleted for eif3e (int6, yin6, SPBC646.09c; h- int6::kanMX6 ura4-D18 leu1-32). Following rRNA depletion and library construction, samples were sequenced on the SOLID4 platform. Two replicates were analyzed and statistical analysis was performed to identify significant differences in mRNA levels.

2016-07-29 | E-GEOD-80349 | biostudies-arrayexpress

Cottus

Project description:Calibration of a custom designed microarray

| PRJNA156917 | ENA