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| Status |
Public on May 31, 2019 |
| Title |
Δlmo01408 replicate2 |
| Sample type |
SRA |
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| Source name |
bacterial cells
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| Organism |
Listeria monocytogenes EGD-e |
| Characteristics |
genotype: [delta]lmo1408
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| Treatment protocol |
25 ml of the culture was collected and quenched by adding of 25 ml ice cold killing buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl2, 20 mM NaN3). After 5 min incubation on ice, cells were harvested by centrifugation.
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| Growth protocol |
Strains were grown in BHI broth to an OD600 of ~0.5 (mid exponential phase).
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction followed the protocol of Völker et al. 1995 (01 April 1994, Microbiology 140: 741-752, doi: 10.1099/00221287-140-4-741) with modifications. Cell pellets were resuspended in 1 ml Lysis Buffer I (25% sucrose; 20 mM Tris-HCl pH 8, 0.25 mM EDTA) and 2 µl of lysozyme (100 mg/ml) was added. After incubation for 5 min on ice, the samples were pelleted (15,000 x g for 30 s at 4°C) and resuspended in 300 µl Lysis Buffer II (3 mM EDTA, 200 mM NaCl). This solution was then mixed with 300 µl Lysis Buffer III (3 mM EDTA, 200 mM NaCl, 1% SDS) that had been pre-incubated at 95°C and the mixture was incubated at 95°C for five more minutes. 600 µl phenol/chloroform/isoamylalcohol (25:24:1) was added and vigorously mixed at room temperature for 5 min. Phases were separated by centrifugation (15,000 x g, 5 min), the aqueous upper phase was collected and the phenol/chloroform extraction was repeated. Afterwards, the aqueous phase was mixed with 600 µl chloroform/isoamylalcohol (25:1), shaken vigorously for 5 min and phases were separated by centrifugation (5 min at 15,000 x g). The chloroform extraction was repeated. Finally, RNA was precipitated by addition of 0.1 x volume 3 M sodium acetate (pH 5.2) and 1.5 volumes 96% ethanol and incubation at -20°C overnight and pelleted by centrifugation (15,000 x g for 15 min at 4°C). The pellet was washed with 70% ethanol and resuspended in 100 µl DEPC treated water. 10 µg total RNA were digested with DNAse using the RNase-Free DNase Set (Qiagen). RNA was then purified using RNA clean & concentrator columns (Zymo Research) for purification for RNA molecules longer than 200 nucleotides. RNA quality was assessed using Agilent Bioanalyzer RNA Nano chips.
rRNA depletion was performed using the Ribo-Zero Bacteria Kit (Illumina) following the manufacturer's instructions. A total of approximately 2 µg purified RNA was trestedtreated with 10 µl Ribo-ZeroRemoval Solution and RNA concentrations were determined using a Qubit® fluorometer. After rRNA removal the remaining RNA was pelleted by ethanol precipitation following the recommendations in the Ribo-Zero protocol. RNA libraries were prepared using the TruSeq® Stranded mRNA Kit, starting with 19.5 µl Fragment, Prime, Finish Mix being added to the dried RNA pellet after rRNA depletion and ethanol precipitation. From this point on the manufacturers protocol was strictly followed (Illumina Part # 15031047 Rev. E). The RNA libraries were sequenced in paired-end mode with 2 times 76 cycles on the Illumina MiSeq (MiSeq® Reagent Kit v3; 150 cycles).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
depletion of bacterial rRNA using Illumina Ribo-ZeroTM
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| Data processing |
Quantification of transcript abundancy via quasi-mapping of raw reads using salmon-0.8.2_linux_x86_64 (https://github.com/COMBINE-lab/salmon)
For statistical significance a two-sided t-test was performed on log-transformed TPM values obtained from salmon output (quant.sf files) assuming heteroscedastic data
Significant hits were selected as having p < 0.01, an expression ratio > 2 and at least 10 TPM detected in all three replicates for wildtype or mutant
Genome_build: L. monocytogenes EGDe cDNA (ftp://ftp.ensemblgenomes.org/pub/bacteria/release-38/fasta/bacteria_0_collection/listeria_monocytogenes_egd_e/cdna/Listeria_monocytogenes_egd_e.ASM19603v1.cdna.all.fa.gz)
Supplementary_files_format_and_content: The *_quant.sf files contain the quantification output of salmon as tab delimited txt, the csv file contains a summary of the raw data as well as the statistics calculated from it.
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| Submission date |
Apr 16, 2019 |
| Last update date |
Jun 01, 2019 |
| Contact name |
Samuel Hauf |
| E-mail(s) |
[email protected]
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| Organization name |
Robert Koch-Institute
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| Department |
Division of Enteropathogenic Bacteria and Legionella (FG11)
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| Street address |
Burgstr. 37
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| City |
Wernigerode |
| ZIP/Postal code |
38855 |
| Country |
Germany |
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| Platform ID |
GPL19076 |
| Series (1) |
| GSE129904 |
Transcriptomic comparison of Listeria monocytogenes EGDe and its Δlmo1408 mutant. |
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| Relations |
| BioSample |
SAMN11439840 |
| SRA |
SRX5696398 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3724883_replicate2_LMSH1_quant.sf.txt.gz |
33.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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