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Using RNA Seq to validate transcriptional profile data obtained by Nanostring analysis

ABSTRACT: Purpose : The goal of this study was to use RNA Seq to validate transcriptional data of two clinical isolates focussing on a subset of 74 transcript that were selected specifically for Nanostring analysis. Methods : mRNA profiles were generated for the clinical isolates FRD1 and CI224_M, in duplicate, by deep sequencing. Strains were grown for 8 hours in LB medium at 37C prior to RNA harvest. Ribosomal RNA was removed using the Ribi-Zero rRNA Removal Kit (Epicentre). mRNA reads were trimmed and mapped to the PAO1 NC_002516 reference genome from NCBI using the ClC Genomics Workbench platform and defaut parameters.

ORGANISM(S): Pseudomonas aeruginosa

PROVIDER: GSE83773 | GEO | 2016/08/09

SECONDARY ACCESSION(S): PRJNA326956

REPOSITORIES: GEO

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Using RNA Seq to validate transcriptional profile data obtained by Nanostring analysis

Project description:Purpose : The goal of this study was to use RNA Seq to validate transcriptional data of two clinical isolates focussing on a subset of 74 transcript that were selected specifically for Nanostring analysis. Methods : mRNA profiles were generated for the clinical isolates FRD1 and CI224_M, in duplicate, by deep sequencing. Strains were grown for 8 hours in LB medium at 37C prior to RNA harvest. Ribosomal RNA was removed using the Ribi-Zero rRNA Removal Kit (Epicentre). mRNA reads were trimmed and mapped to the PAO1 NC_002516 reference genome from NCBI using the ClC Genomics Workbench platform and defaut parameters. mRNA profiles of liquid cultures grown for 8 hours in LB at 37C were generated for P. aeruginosa clinical isolates FRD1 and CI224_M, each in duplicate, by deep sequencing using Illumina NextSeq.

2016-08-09 | E-GEOD-83773 | biostudies-arrayexpress

Using RNA Seq to define the regulon of the Pseudomonas aeruginosa transcription factor Anr in low-oxygen colony biofilms.

Project description:Purpose : The goal of this study was to use RNA Seq to define the regulon of the transciption factor Anr by comparing global transcriptional profiles of Pseudomonas aeruginosa strain PAO1 and a clinical isolate with their isogenic ?anr mutants, grown in colony biofilms at 1% oxygen. Methods : mRNA profiles were generated for laboratory strain PAO1 and for a clinical isolate J215, as well as for ?anr derivatives of each strain, in duplicate, by deep sequencing. Strains were grown for 12 hours in colony biofilms at 1% O2, 5% CO2 prior to RNA harvest. Ribosomal and transfer RNAs were removed using the MICROBExpress kit (Life Technologies). mRNA reads were trimmed and mapped to the PAO1 NC_002516 reference genome from NCBI using the ClC Genomics Workbench platform and defaut parameters. mRNA profiles of 12 hour colony biofilms were generated for P. aeruginosa strains PAO1 WT, PAO1 ?anr, clinical isolate J215, and J215 ?anr, each in duplicate, by deep sequencing using Illumina HiSeq.

2015-06-29 | E-GEOD-68534 | biostudies-arrayexpress

Using RNA Seq to define the regulon of the Pseudomonas aeruginosa transcription factor Anr in low-oxygen colony biofilms.

Project description:Purpose : The goal of this study was to use RNA Seq to define the regulon of the transciption factor Anr by comparing global transcriptional profiles of Pseudomonas aeruginosa strain PAO1 and a clinical isolate with their isogenic ∆anr mutants, grown in colony biofilms at 1% oxygen. Methods : mRNA profiles were generated for laboratory strain PAO1 and for a clinical isolate J215, as well as for ∆anr derivatives of each strain, in duplicate, by deep sequencing. Strains were grown for 12 hours in colony biofilms at 1% O2, 5% CO2 prior to RNA harvest. Ribosomal and transfer RNAs were removed using the MICROBExpress kit (Life Technologies). mRNA reads were trimmed and mapped to the PAO1 NC_002516 reference genome from NCBI using the ClC Genomics Workbench platform and defaut parameters.

2015-06-29 | GSE68534 | GEO

Identification of PUMPKIN RNA targets using RIPSeq

Project description:Stroma extracts were isolated from 2-week-old WT plants and incubated with either PUMPKIN-specific antibodies or with the pre-immune serum. IgGs were captured with SiMAG-Protein G beads (Chemicell) and recovered RNA was used for generation of libraries with the ScriptSeq v2 RNA-seq Library Preparation Kit (Epicentre). Primary reads were aligned to the Arabidopsis chloroplast genome (accession number NC_000932.1) using CLC Genomics Workbench, the mean RPKM values of the replicates as well as the ratio of PUMPKIN vs Pre-immuneserum were calculated. Five prominent RNA targets (trnG-UCC, trnV-UAC, petB, petD and ndhA) were identified and validated via Slot Blot analyses.

2019-10-01 | GSE121777 | GEO

PAHM4 vs PAO1 37C LB

Project description:PAHM4 vs PAO1 37C LB

| PRJNA174088 | ENA

Identification of BSF RNA targets using RIPSeq

Project description:Stroma extracts were isolated from 2-week-old WT plants and incubated with either BSF-specific antibodies or with the pre-immune serum. IgGs were captured with SiMAG-Protein G beads (Chemicell) and recovered RNA was used for generation of libraries with the ScriptSeq v2 RNA-seq Library Preparation Kit (Epicentre). Primary reads were aligned to the Arabidopsis chloroplast genome (accession number NC_000932.1) using CLC Genomics Workbench. BAM files were extracted and sorted in Galaxy . Sorted BAM files were converted into RPKM-normalized bigwig files and displayed in IGB. The differential enrichment of BSF/control of the two replicates was displayed across the entire chloroplast genome to identify the RNA targets of BSF.

2019-04-06 | GSE129411 | GEO

Next-Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Monocyte-Depleted Brain Transcriptomes

Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived brain transcriptome profiling (RNA-seq) to reveal the difference of cellular pathways in adult brains of wild type (WT) and monocyte-depleted (CCR2-DTR) mice 8 days post infection of Trichinella spiralis. Methods: Illumina compatible RNAseq library was prepared using NEB next ultra RNAseq library preparation kit. The sequencing of the cDNA libraries was performed on the Illumina NextSeq platform (Illumina, San Diego, CA) using the high output 1X75 cycles configuration. CLC Genomics Workbench 11.0.1 version (http://www.clcbio.com/products/clc-genomics-workbench/ ; Qiagen) was used for RNA-seq analysis. De-multiplexed fastq files from RNA-Seq libraries are imported into the CLC software. Bases with low quality were trimmed and reads are mapped to reference genome Mus musculus genome GRCm38. The reference genome sequence and annotation files were downloaded from ENSEMBLE, release.92 (Mus_musculus.GRCm38.92.fa, and Mus_musculus.GRCm38.92.gtf). The aligned reads were obtained using the RNA-Seq Analysis Tool of CLC Genomics Workbench. Statistical analysis of differentially expressed genes is carried out based on a negative binomial model using a tool in CLC Genomic Workbench. The reference genome sequence and annotation files were downloaded from ENSEMBL, release.92 (Mus_musculus.GRCm38.92.fa, and Mus_musculus.GRCm38.92.gtf). The aligned reads were obtained using the RNA-Seq Analysis Tool of CLC Genomics Workbench. Results: Using an optimized data analysis workflow, we mapped over 50 million sequence reads per sample to the mouse genome (GRCm38) and found that Trichinella-infected mice depleted of monocytes presented with significantly increased expression of proinflammatory genes compared to controls.

2022-08-25 | GSE190493 | GEO

Conformation/Identification of dPPRrcbL RNA targets using RIPSeq

Project description:Stroma extracts were isolated from 2-week-old transgenic dPPRrbcL or WT plants and incubated with HA-specific antibodies. IgGs were captured with Protein A Dynabeads (Thermo Fisher scientific) and recovered RNA was used for generation of libraries with the NEBNext® Ultra™ II RNA Library Prep Kit. Primary reads were aligned to the Arabidopsis chloroplast genome (accession number NC_000932.1) using CLC Genomics Workbench. Reads of the two replicates aligned in CLC Genomics Workbench were extracted as coverage (reads per nucleotide). Graphs were created with excel using the extracted coverage values

2021-02-28 | GSE146249 | GEO

PAHM4 vs PAO1 37C LB

Project description:Pseudomonas aeruginosa strains PAHM4 and PAO1 were grown at 37C on LB and RNA was hybridized on the Affymetrix P. aeruginosa chip to compare transcript differences from a BQ isolate to a well characterized wound isolate.

2012-10-01 | GSE40461 | GEO

CARD9-associated Dectin-1 and Dectin-2 are Required for Protective Immunity of a Multivalent Vaccine Against Coccidioides posadasii Infection

Project description:Preparation of sequencing libraries and sequencing analysis using Illumina HiSeq 3000 platform (50 bp single-read module) were conducted at the Genomics Core of UT Health San Antonio. Sequencing reads were preprocessed by Trim Sequences tools in CLC Genomics workbench. Remaining reads were then aligned to mouse GRCm38(mm10) reference genome using RNA-Seq Analysis module in CLC Genomics workbench. To determine differentially expressed genes, exact test were performed after filtering lowly expressed gene (counts per million < 1 in more than three samples across the dataset) using edgeR package (Robinson, McCarthy et al. 2010). Gene Ontology (GO) analysis were performed using DAVID (Dennis, Sherman et al. 2003) and then visualized by GOplot (Walter, Sanchez-Cabo et al. 2015).

2020-04-16 | GSE133140 | GEO

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