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| Status |
Public on Nov 05, 2017 |
| Title |
MT02_128-Rep_1 |
| Sample type |
SRA |
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| Source name |
bacterial cells
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| Organism |
Staphylococcus aureus |
| Characteristics |
background strain: LAC USA300 JE2
mt02 sensitivity: resistant
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| Treatment protocol |
MT02-resistant S. aureus was incubated in the presence of MT02 (80 µM). RNA was isolated from the cells at OD600= 1. At least 6 mL of the bacterial cultures were harvested by centrifugation for 10 min at 10,000 rpm, and the bacterial pellet was resuspended in 800 µL of RLT buffer (Qiagen, Hilden, Germany) and mechanically disrupted with glass beads (2 mL Lysing Matrix tubes, MP Biochemicals GmbH, Eschwege, Germany) in a Fastprep®-24 (MP Biochemicals). The cell lysate was centrifuged for 2 min at 13,000 rpm, and the supernatant was used for RNA isolation.
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| Growth protocol |
Bacterial strains (MT02-sensitive S. aureus JE2 and S. aureus JE2 strain resistant to 80 µM MT02) were cultivated overnight at 37°C in LB medium in a shaking incubator at 200 rpm. The next day, the strains were diluted in 100 mL MH broth (optical density was adjusted at OD600= 0.05) and again incubated until the cells reached the exponential growth phase.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated with an RNeasy Mini kit (QIAGEN) according to the instructions of the manufacturer. To remove the DNA template, RNA was treated with RNase-free DNase I (New England, Biolabs® Inc, Frankfurt am Main, Germany).
Total RNA of MT02-resistant and wild-type strain JE2 was extracted with the RNeasy (Qiagen) or the MirVana isolation kit (Thermo Fisher Scientific). Fragmentation of messenger-enriched RNA was performed using zinc. Specific tags were ligated before reverse transcription to conserve strand orientation. Single-stranded ligation of the 3′ Illumina adapter was followed by single-stranded ligation of 5′ bar-coded adapter. Reverse transcription and PCR amplification were performed to generate a cDNA colony template library, and gel purification with selection of fragments of 20 to 300 nt was performed. For HTS experiments, total RNA was treated with the MICROBExpress kit (Thermo Fisher Scientific) to limit contamination by multicopy structural rRNAs, following manufacturer's instructions with slight modifications. The maximal amount of total RNA per tube was limited to 4–5 µg because previous experiments showed important contamination levels when using 10 µg. Between each purification step, RNA quality and quantity were determined by Bioanalyzer (Agilent) using the RNA Nano chips, and quantified using the ND-8000 (Thermo Fisher Scientific).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
FastQ quality trimming: cut-off value of 20 (FastX 0.0.13)
Adaptor trimming (cutadapt 1.10)
Read mapping (READemption 0.4.2 and segemehl version 0.2.0)
Wiggle generation and gene quantification (READemption 0.4.2)
Genome_build: NC_007790.1, NC_007791.1, NC_007792.1, NC_007793.1
Supplementary_files_format_and_content: wiggle
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| Submission date |
Oct 03, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Konrad U. Förstner |
| E-mail(s) |
foerstner@zbmed.de
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| Organization name |
ZB MED - Information Centre for Life Sciences
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| Department |
Information Services
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| Lab |
Förstner Lab
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| Street address |
Gleueler Str. 60
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| City |
Cologne |
| State/province |
North Rhine-Westphalia |
| ZIP/Postal code |
50931 |
| Country |
Germany |
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| Platform ID |
GPL16057 |
| Series (1) |
| GSE87572 |
RNA-Seq based comparison of Staphylococcus aureus strains resistent and sensitive to MT02 |
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| Relations |
| BioSample |
SAMN05859649 |
| SRA |
SRX2208173 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2334638_MT02_128-Rep_1_forward.wig.gz |
5.3 Mb |
(ftp)(http) |
WIG |
| GSM2334638_MT02_128-Rep_1_reverse.wig.gz |
5.4 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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