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| Status |
Public on Jun 13, 2017 |
| Title |
19C |
| Sample type |
SRA |
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| Source name |
aerated pure culture replicate C
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| Organism |
Lactococcus garvieae |
| Characteristics |
lactococcus garvieae strain: N201
culture type: pure culture
aeration level: high
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| Treatment protocol |
conservation at -80°C
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| Growth protocol |
Both strains were inoculated separately or in co-culture at 1E+06 cells.mL-1 for S. aureus or 1E+07 cells/mL for L. garvieae in BHI buffered at pH = 7 with phosphate buffer KH2PO4, 3H2O/K2HPO4 (KH2PO4, 3H2O, Riedel-de-Haen, Honeywell GmbH, Seelze, Germany; K2HPO4, Merck KGaA, Darmstadt, Germany) previously equilibrated at 30°C. Pure cultures and co-cultures of both strains were performed either under a high or under a low aeration level depending on the experiment. Low aeration level cultures were set in static fully filled and sealed 50-mL Nunc EZ Flip conical centrifuge tubes (Sigma-Aldrich, St. Louis, Missouri, USA). The high aeration level was obtained with a mechanical shaking at 150 rpm on 50-mL cultures in 250-mL erlenmeyers. All cultures were incubated at 30°C for 24h in an Infors HT Minitron (Infors AG, Bottmingen, Switzerland). After 9 h, 40 mL of each culture were centrifuged at 9,600 x g for 10 min at 4°C. The cell pellets were immediately frozen in an ethanol bath and then stored at -80°C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen cell pellets obtained from cultures at 9 h were suspended in 500 µL of cold Tris-EDTA buffer. Then, 25 µL of 20% SDS, 500 µL of phenol at pH 4, 3.5 µL of β-mercaptoethanol, and 600 mg of Zirconium beads were added. The solution was milled in a Retsch MM200 mixer mill (VERDER Group, Haan, Germany) at 30 hertz for two 2-minute runs with 2-minute cooling on ice in between. Next, 200 µL of chloroform were added. The solution was gently hand-mixed and centrifuged at 13,000 x g for 20 min at 4°C. Extraction of RNAs was then performed on the aqueous and translucent phase using NucleoSpin RNA-L or RNA-II kit (Macherey-Nagel, GmbH & Co. KG, Düren, Germany) following the suppliers instructions. Then we treated RNA extracts with rDNAse I using an Ambion DNA-free™ Kit (Ambion, Inc., Austin, Texas, USA) following the suppliers instructions. Absence of residual DNA was confirmed by qPCR with S. aureus gyrB gene primers. If needed, rDNAse I treatment was repeated until Cycle threshold (Ct) were > 30. The RNA extracts were quantified using a Nanodrop™ 2000C (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA). Ribosomal RNAs were depleted from the total RNA (2 x 5 µg of RNA by sample) using a Magnetic Kit RiboZero for Gram Positive Bacteria (Illumina Inc., San Diego, California, USA) according to the manufacturer’s instructions. The quality and concentration of RNA in each sample were assessed using a RNA 6000 pico kit (Agilent Technologies, Santa Clara, California, USA).
RNA libraries were prepared by the MGX Plateform (Montpellier GenomiX, CNRS, Montpellier, France) using Illumina kit (TruSeq Stranded mRNA Sample Preparation kit), devices and standard protocols (Illumina Inc.)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
RNA after rRNA depletion
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| Data processing |
Basecalls performed using RTA v 1.9 (Illumina)
Concatenation of 4-5 fastq per samples
RNA seq were aligned on both genomes (S.aureus MW2+L.garvieae N201) using BWA (v0.7.5a-r405) aln with parameters -l 32 -k 2, BWA samse, samtools view -h -s -q 20 to suppress low quality alignment (to visualize alignements: … samtools view -bt to compress sam into bam files, samtools sort and index to obtain final alignment files)
Counting aligned reads: Htseq version 0.5.4p5 (i) with parameters –stranded=reverse –mode=union –type=CDS –idattr=ID for L. garvieae reads counting (ii) with parameters --stranded=reverse --mode=union --type=CDS –idattr=Dbxref for S. aureus reads counting
Differential expression analysis is carried out on count files for each conditions with R-packages DESeq, DESeq2, EdgeR, R v3.1.1
Genome_build: Lactococcus garvieae N201 (GOLD project ID: Gp0034836); Staphylococcus aureus MW2 (NCBI Reference Sequence: NC_003923)
Supplementary_files_format_and_content: Tab-delimited text files (file name = condition1_condition2_R-package used.txt) list only L. garvieae CDSs significatively differentially expressed (condition 1/condition 2) with log(fold-change) and p-values (raw and ajusted). Files are available in a tar archive located on the series record.
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| Submission date |
Oct 14, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Pierre DELPECH |
| E-mail(s) |
[email protected]
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| Phone |
+33471456418
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| Organization name |
INRA
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| Department |
MICA
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| Lab |
URF
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| Street address |
20 côte de Reyne
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| City |
AURILLAC |
| State/province |
Auvergne Rhône Alpes |
| ZIP/Postal code |
15000 |
| Country |
France |
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| Platform ID |
GPL21033 |
| Series (1) |
| GSE74030 |
Analysis of the transcriptome of Lactococcus garvieae in presence or absence of Staphylococcus aureus and under a low or a high level of aeration by RNA sequencing |
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| Relations |
| BioSample |
SAMN04166623 |
| SRA |
SRX1336116 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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