Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Exploring copper toxicity mechanisms in Magnetospirillum gryphiswaldense MSR1 using transcriptomics

ABSTRACT: This experiment investigated the transcriptional response of the magnetotactic bacterium Magnetospirillum gryphiswaldense MSR1 to acute copper stress. Mid-exponential-phase cultures were exposed to five milligrams per litre copper for three hours, with untreated cultures sampled at the start and end of the incubation period. Three biological replicates were analysed for each condition. RNA was extracted, sequenced and subjected to quality assessment before reads were mapped to the MSR1 reference genome and gene counts were analysed using DESeq2. The primary comparison assessed copper-exposed cultures against time-matched controls, while the control time comparison provided context for normal growth-associated transcriptional changes.

INSTRUMENT(S): incubator shaker; spectrophotometer; Cmag measurement system; flow cytometer, thermal cycler; magnetic separation rack; Qubit Fluorometer; Agilent TapeStation 4200, Element AVITI, microcentrifuge; magnetic separation rack; Qubit Flu/spectrophotometer; Agilent TapeStation 4200

ORGANISM(S): Magnetospirillum gryphiswaldense

SUBMITTER: Josh Bond

PROVIDER: E-MTAB-17612 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:RNA-seq analysis was performed to understand the role of type I IFN response during SARS CoV-2 infection using transgenic mice. Each sample was collected from an individual C57BL/6J mouse. The total RNA was extracted from uninfected and SARS-CoV-2 infected mice lung tissue using RNeasy mini kit (QIAGEN #74104). The quantity of RNA was determined using Qubit RNA assay kit with Qubit 4.0 and the quality of RNA was tested using agarose gel electrophoresis and High Sensitivity Tape station Kit (Agilent 2200, #5067-5576, #5067-5577 and #5067-5578). After assessing the quality of RNA, ~900 ng of total RNA was taken for library preparation using NEBNext®Ultra™ II Directional RNA Library kit for Illumina (# E7760L) and NEBNext Poly (A) mRNA Magnetic Isolation Module (# E7490L) as per manufacturer's protocol. The prepared library was quantified using Qubit dsDNA assay kit (Invitrogen, Q32851) followed by quality check (QC) and fragment size distribution using a High Sensitivity Tape station Kit (Agilent 2200, #5067-5584 and #5067-5585). The library was sequenced using the HiSeq 4000 Illumina platform. The paired-end (PE) reads quality checks for each sample were carried out using FastQC v.0.11.5 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The adapter sequence was trimmed using the BBDuk version 37.58 version 37.58 and the alignment was performed using STAR v.2.5.3a with default parameters with human hg38 genome build, gencode v21 gtf 9GRCh38) from the gencode. The duplicates were discarded using Picard-2.9.4 (https://broadinstitute.github.io/picard/) from the aligned bam files and read counts were generated using featureCount v.1.5.3 from subread-1.5.3 package (https://bioinf.wehi.edu.au/) with Q = 10 for mapping quality. The count files were used as input for downstream differential gene expression analysis with DESeq2 version 1.14.1 9. The genes with read counts of ≤ 10 in any comparison were discarded followed by count transformation and statistical analysis using DESeq “R”. The “P” value were adjusted using the Benjamini and Hochberg multiple testing correction and the differentially expressed genes were identified (fold change of ≥1.5, P-value < 0.05). A unified non-redundant gene list was made for different comparisons and subjected to gene ontology (GO) analysis using the reactome database (https://reactome.org/). The top pathways (p < 0.05) were used for generating heat maps using Complexheatmap (Version 2.0.0) through unsupervised hierarchical clustering. The expression clusters were annotated based on enriched GO terms. Normalized gene expression was used to generate the boxplots with a median depicting the trends in the expression across the different conditions using ggplot2 [version 3.3.5]. The pathways analysis was performed using Metascape database (https://metascape.org/gp/index.html#/main/step1). The top pathways (p < 0.05) were taken for constructing bubble plots using ggplot2 [version 3.3.5].

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Exploring copper toxicity mechanisms in Magnetospirillum gryphiswaldense MSR1 using transcriptomics

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