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Sample GSM2089716

Query DataSets for GSM2089716

Status

Public on Mar 16, 2016

Title

AgXX.t06

Sample type

SRA

Source name

Cell

Organism

Enterococcus faecalis

Characteristics

strain: 12030
growth in the presence of steel mesh: AGXX
exposure time [min]: 6

Treatment protocol

E. faecalis 12030 cultures in mid-exponential growth phase (OD600 ≈ 0.6) were either subjected to metal stress by exposure to an AGXX®- or Ag-coated V2A steel mesh or exposed to an uncoated V2A steel mesh (12 cm2 each in 30 ml BHI medium to obtain a mesh-surface : medium-volume ratio of 0.4) followed by further incubation for 3, 6, 12, 24, 60 and 90 min at 37°C with constant agitation at 150 rpm. As control, no metal mesh was added.

Growth protocol

Enterococcus faecalis 12030 was pre-cultured overnight at 37°C in Brain Heart Infusion (BHI, Oxoid Deutschland GmbH, Wesel, Germany) medium with constant agitation at 150 rpm and diluted in BHI medium to an OD600 of 0.05.

Extracted molecule

total RNA

Extraction protocol

Cells from 30 ml culture were harvested by centrifugation for 1 min at 10 000 rpm and 4°C in a Sorvall RC6+ centrifuge (Thermo Scientific GmbH, Langenselbold, Germany). Cell pellets were immediately frozen in liquid nitrogen and stored at -80°C or directly used for RNA isolation. RNA was isolated using the ZR Fungal/Bacterial RNA MiniPrepTM Kit (Zymo Research, Freiburg, Germany) following the manufacturer's instructions. To recover total RNA including small RNAs, 1.5 volumes of absolute ethanol were added in step 5. Finally, total RNA was eluted with 50 µl DNase-/RNase-free water and stored at -80°C. RNA quantity and quality were assessed with Agilent RNA 6000 Nano and Pico Kits using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Residual contaminating DNA was digested with DNA-freeTM DNase (Ambion, Carlsbad, USA).
To remove rRNAs and tRNAs from the total RNA, MICROBExpressTM and MEGAclearTM Kits were applied. After purification, 500 ng of the enriched (m)RNA was fragmented with RNase III. Fragmented (m)RNA (50-100 ng) was used for the preparation of barcoded whole transcriptome cDNA libraries using the Ion Total RNA-Seq Kit v2 and Ion XpressTM RNA-Seq Barcode 1-16 Kits. Finally, strand-specific, multiplexed RNA sequencing was carried out on Ion PITM chips in an Ion ProtonTM Sequencer. All kits and devices for RNA sequencing were from Ambion, Carlsbad, USA and used according to the manufacturer's instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Ion Torrent Proton

Data processing

Raw sequencing reads were aligned to the reference genome of E. faecalis 12030 using Bowtie2 (version 2.2.3).
Post-processing of the SAM files into sorted BAM files was performed with SAMtools (version 1.2-207).
Length normalized confidence interval Fragments Per Kilobase of exon per Million fragments mapped (FPKM) values were obtained with Cufflinks using optimized settings for the Ion ProtonTM Sequencer.
Statistical analysis was carried out with the recently developed T-REx RNA sequencing expression analysis pipeline (de Jong et al., BMC Genomics, 2015) using the differential expression method of EdgeR.
A gene was considered significantly differentially expressed when the expression ratio was ≥|2.0| and the false discovery rate (FDR) adjusted p-value ≤ 0.05.
Genome_build: Enterococcus faecalis 12030
Supplementary_files_format_and_content: Tab-delimited txt files with GeneID, logFC, logCPM, LR, pvalue, adj_pvalue and Fold value for each contrast (= comparison of sample X versus sample Y).
Supplementary_files_format_and_content: Tab-delimited txt file with FPKM for all samples.

Submission date

Mar 15, 2016

Last update date

May 15, 2019

Contact name

Emanuel Clauss-Lendzian

E-mail(s)

e.clauss-lendzian@online.de

Organization name

University Medical Center

Street address

Hugstetter Straße 55