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| Status |
Public on Jun 01, 2016 |
| Title |
SXT 3h BIO2 |
| Sample type |
SRA |
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| Source name |
Listeria monocytogenes_SXT_3h
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| Organism |
Listeria monocytogenes |
| Characteristics |
strain: EGD
treatment: 0.2 µg/ml co-trimoxazole at time 3h
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| Growth protocol |
To obtain balanced growth, an overnight culture of Listeria monocytogenes EGD was diluted 10^9-fold in 5 ml BHI and grown for 16h at 37˚C at 250 rpm. This culture was used for inoculation of 50 ml prewarmed BHI to OD600 = 0.01 and grown at 37˚C with 250 rpm to OD600 = 0.1. The culture was split in five by diluting 8 ml culture with 42 ml pre-warmed BHI broth and incubated again. At OD600 = 0.1, equal volumes of antibiotic or MQ water were added to each flask to obtain final concentrations of 0.03 µg/ml ampicillin, 0.035 µg/ml tetracycline, 0.3 µg/ml gentamicin or 0.2 µg/ml co-trimoxazole. Samples for gene expression and bacterial counts were taken at time zero of supernatant addition for the MQ control and at 3h for all samples, with two independent biological replicates.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Samples for RNA sequencing were harvested from antibiotic-exposed and control cells at 0h and 3h and were quenched for 30 min in ice-cold 2% phenol, 38% ethanol, 62% water, to stabilise RNA (Eriksson et al., 2003). Cells were pelleted by centrifugation and stored at -80°C until total RNA extraction using Trizol (Invitrogen) according to Gomez-Lozano et al. (2012). Quantity and quality of the purified total RNA was evaluated by Nanodrop and Agilent 2100 Bioanalyzer using the RNA 6000 Nano kit. To reduce the amount of rRNA reads for the RNA sequencing, we isolated mRNA from good quality total RNA (RIN 9.8-10) using the MICROBExpress kit (Ambion #AM1905) according to the manufacture’s protocol. Depletion of the 16S and 23S was verified on a RNA 6000 Nanochip (2100 Bioanalyser, Agilent) and quantity was measured using a Qubit RNA assay (Invitrogen).
Libraries for RNA Seq were prepared using the 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 325 ng mRNA, which was mixed with 13 µl Elute, Prime and Fragment mix and fragmented to approximately 180 bp. Specific adaptors were ligated to each of the 20 samples. The library was validated by measuring quantity with the Qubit dsDNA BR assay kit and quality with the 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 10.3 nM with an average base pair size of 270.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
mRNA and sRNAs
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| 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 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 statistically significant gene expression differences with a statistical filter of fold changes above 2, P-value < 0.05 and a false discovery rate (FDR) of < 0.05
Genome_build: 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
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| Submission date |
Feb 03, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Gitte M. Knudsen |
| E-mail(s) |
gmkn@bio.dtu.dk
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| Organization name |
Technical University of Denmark
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| Department |
Department of Systems Biology
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| Lab |
BEB
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| Street address |
Matematiktorvet, Bldg 301
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| City |
Kgs. Lyngby |
| State/province |
Not applicable |
| ZIP/Postal code |
2800 |
| Country |
Denmark |
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| Platform ID |
GPL17257 |
| Series (1) |
| GSE65558 |
Sublethal concentrations of antibiotics cause shift to anaerobic metabolism in Listeria monocytogenes and induce phenotypes linked to antibiotic tolerance |
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| Relations |
| BioSample |
SAMN03326273 |
| SRA |
SRX864210 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1600073_Index21_GTTTCG_GEN_3h_BIO3.txt.gz |
69.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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