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Sample GSM1246399 Query DataSets for GSM1246399
Status Public on Jan 31, 2014
Title 0.1875% 180min rep2
Sample type SRA
Source name 0.1875% 180min rep2
Organism Listeria monocytogenes EGD
Characteristics protocol: 0.1875% L. plantarum WHE 92 supernatant
strain: EGD
Growth protocol An overnight L. monocytogenes EGD BHI (Brain Heart Infusion) culture was diluted 100-fold in prewarmed BHI and incubated at 37°C with 250 rpm. When the culture reached OD600nm ≈ 0.4, the culture was split into four, diluting the culture four times in prewarmed BHI and reincubated at 37°C with 250 rpm. At OD600nm ≈ 0.4, equal volumes of L. plantarum WHE 92 supernatant or MQ were added each flask to obtain final concentrations of 0% (MQ control), 0.125%, 0.1875% and PT 0.1875% supernatant. Samples for gene expression and bacterial counts were taken at time zero of supernatant addition, at 10 min, 60 min and 180 min with two independent biological replicates.
Extracted molecule total RNA
Extraction protocol RNA samples were stabilized with RNAprotectTM (Qiagen 76506) and stored at – 80°C prior to RNA purification with RNeasy Mini Kit (Qiagen 74104) including DNase treatment with RNase free DNase (Qiagen 79254) according to the manufacturer’s instructions. Quantity and quality of the purified total RNA was evaluated by Nanodrop and Agilent 2100 Bioanalyzer using the RNA 6000 Nano kit. Five µg total RNA from each sample was ethanol precipitated, dissolved in 15 ul RNase free water and used as input in MICROBExpressTM Kit (Ambion AM1905) to remove of 16S and 23S rRNA. mRNA samples were evaluated on Qubit RNA assay kit (Invitrogen Q32852) and on Agilent 2100 Bioanalyzer with the RNA 6000 Nano kit.
Libraries for RNA Seq were prepared using TruSeq RNA Sample preparation Kit v2 (set A: RS-122-2001 and set B: RS-122-2002) according to the manufactures protocol. In brief, cDNA was generated from mRNA, which had been fragmented to approximately 180 bp. Specific adaptors were ligated to each of the 20 samples. The library was validated by measuring quantity with Qubit dsDNA BR assay kit and quality with Agilent 2100 Bioanalyzer on DNA-1000 chip. Due to a peak at 1500 bp, an E-gel size select (Invitrogen G6610-02) was performed to isolate the cDNA sequences of 200-400 bp. Lastly, each library was normalized to 10 nM and pooled yielding a final concentration of 7.92 nM with an average base pair size of 290.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
Data processing The RNA seq data was analyzed using the CLC genomics workbench software (CLC Aarhus, version 6.0). Quality and quantity of RNA sequence data was evaluated by the number of reads, GC%, PHRED-score, nucleotide contribution, quality distribution and enriched 5mers sequences. All reads were trimmed removing the 15 first nucleotides from the 5´end due to non-normal nucleotide distribution. Subsequently, reads were sorted according to their respective adaptor sequences and individually aligned to the reference genome. Reads Per Kilobase per Million (RPKM) was used as expression value.Quality control of the transcriptomic data was evaluated by use of hierarchal clustering and principal component analysis. The gene expression profiles of biological replicates were paired and compared between treated and control samples within the same time point. Baggerlys test was used to evaluate statistical significant gene expression differences with a statistical filter of fold changes above 2, P-value < 0.05 and false discovery rate (FDR) < 0.05
Genome_build: ASM19603v1 [L. monocytogenes EGD-e reference genome (NC_003210.1)]
Supplementary_files_format_and_content: xslx files including total gene reads, RPKM values etc. for each sample
Submission date Oct 21, 2013
Last update date May 15, 2019
Contact name Gitte M. Knudsen
Organization name Technical University of Denmark
Department Department of Systems Biology
Street address Matematiktorvet, Bldg 301
City Kgs. Lyngby
State/province Not applicable
ZIP/Postal code 2800
Country Denmark
Platform ID GPL17803
Series (1)
GSE51487 A single exposure to a sublethal pediocin concentration initiates a resistance-associated temporal cell envelope and general stress response in L. monocytogenes
BioSample SAMN02378653
SRA SRX365767

Supplementary file Size Download File type/resource
GSM1246399_Index018_0.1875_3hrs_BIO4.xlsx.gz 208.5 Kb (ftp)(http) XLSX
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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