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| Status |
Public on Mar 31, 2015 |
| Title |
RBL67_co_2 |
| Sample type |
SRA |
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| Source name |
B. thermophilum RBL67 -S. Typhimurium N-15 co-culture
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| Organism |
Bifidobacterium thermophilum |
| Characteristics |
strain: RBL67
fermentation: RBL67-N15 co-culture
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| Growth protocol |
Two sets of fermentations were performed, each set consisting of six fermentations. The first set was composed of three RBL67 mono-cultures and three RBL67-N15 co-cultures. The second set consisted of three N-15 mono-cultures and three N-15-RBL67 co-cultures. Bacteria were cultured in 350 mL scale Sixfors bioreactors (Infors AG, Bottmingen, Switzerland) using 310 mL YCFA medium supplemented with 6 g/L glucose (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). Fermentations were performed for 24 h at 38°C with stirring at 200 rpm. A constant pH of 6.0 was maintained by automated addition of 2.5 M NaOH. Anaerobic conditions were ensured by purging the headspace with CO2. Both, mono- and co-culture fermentations were inoculated with 4 % (v/v) of a 16 h grown pre-culture. Pre-cultures were prepared by twice propagating RBL67 and N-15 in 10 mL YCFA medium in Hungate tubes to adapt the strains to the medium and anaerobic conditions. The pre-cultures were centrifuged (6000 x g, 5 min), washed in 0.1 % peptone water reduced with 0.05 % L-cysteine hydrochloride (VWR International AG, Dietikon, Switzerland) and resuspended in 2 mL peptone water before inoculation to the fermenter.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from culture samples taken after 5 h (N-15) or 5 h (RBL67) for mono-and co-cultures. RBL67 and N-15 mono- and co-culture samples were subjected to different procedures to allow optimal RNA extraction of both RBL67 and N-15. Mono- and co-culture samples of N-15 cultures (20 mL each) were directly transferred to 20 mL 60 % glycerol (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland) at -40°C, kept on ice for 20 min and centrifuged for 15 min (3220 x g, 4°C). The supernatant was discarded and the resulting pellets were immediately frozen at -80°C until RNA extraction. Mono- and co-culture samples of RBL67 cultures were shortly centrifuged (10000 x g, 20 s). The RBL67 mono-culture pellets were resuspended in 400 μl MRS-C and transferred to a pre-chilled screw cap tube, containing 500 mg glass beads (0.1 mm; Biospec Products Inc., Bartlesville, USA), 500 μl chloroform/phenol (1:1, v/v), 30 μl 3 M Na-acetate (pH 5.2) and 30 μl SDS 10 %. The pellet of the RBL67 co-culture was resuspended in 12 mL of RNAprotect® Bacteria Reagent (Qiagen AG, Basel, Switzerland), incubated for 5 min at room temperature and centrifuged again (10000 x g, 20 s). Both samples were then rapidly frozen in liquid nitrogen and stored at -80°C until RNA extraction. Frozen pellets from N-15 samples were resuspended in 200 μl 10 mM Tris-buffer (pH 8.0). Total RNA was extracted using the High Pure RNA isolation kit (Roche Diagnostics, Rotkreuz, Switzerland), according to the manufacturer’s instructions. Total RNA of RBL67 mono- and co-culture samples was extracted using a phenol/chloroform extraction method, followed by a purification using the High Pure RNA isolation kit (Roche Diagnostics). Prior to RNA extraction the sample from the RBL67 co-culture was resuspended in MRS-C medium and transferred to a pre-chilled mix of 500 mg glass beads (Biospec Products Inc.) and TRI Reagent® (Life Technologies Europe BV, Zug, Switzerland). RNA quantity and purity was determined on a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Washington, USA) and RNA integrity was tested with an Agilent 2100 Bioanalyzer (Agilent, Basel, Switzerland). Depletion of ribosomal RNA from 10 μg total RNA was performed using the MICROBExpress™ Bacterial mRNA Enrichment Kit (Life Technologies Europe BV, Zug, Switzerland) according to the manufacturer’s instructions. Additionally, EDTA (1 mM) was added to chelate divalent cations present in the RNA solution.
RNA-sequencing was performed on an Illumina HiSeq 2000 sequencer (Illumina Inc., California, USA) at the Functional Genomics Center Zurich (FGCZ). Libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina) according to the manufacturer’s protocol. The libraries were qualitatively and quantitatively checked using a Qubit® (1.0) Fluorometer (Life Technologies Europe BV, Zug, Switzerland) and a Bioanalyzer 2100 (Agilent, Basel, Switzerland) and were subsequently normalized at 10 nM in Tris-Cl (10mM, pH 8.5) containing 0.1 % Tween20. Cluster generation was performed using the TruSeq SR Cluster Kit v3-cBot-HS (Illumina) using 8 pM of pooled normalized libraries on the cBOT and stranded sequencing of 100 bp was done using the TruSeq SBS Kit v3-HS (Illumina). Each set of samples (n=6) was analyzed in a separate sequencing lane.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina raw data reads (100 bp) were separated by barcode and mapped against the genome of RBL67 (GenBank accession no. CP004346) or Salmonella Typhimurium LT2 (GenBank accession no. AE006468) using CLC Genomics Workbench 6.5.1 (http://www.clcbio.com), applying the default settings. Maximum allowance of mismatches was set at 2, minimum length fraction at 0.9 and minimum similarity fraction at 0.8. The raw data provided are the total counts per gene, not normalized against gene length or library size.
Genome_build: CP004346 (ASM34769v1) and AE006468 (ASM694v1)
Supplementary_files_format_and_content: The processed data files contain a table with in the first column the locus_tag of the gene as it is stored in NCBI Genbank (<locus_tag>), in the second and third column the position on the chromosome(<start><stop>), in the fourth column the strand (<strand>), in the fifth column the length of the encoded protein in amino acids (<length (AA)>), and the sixth column the protein ID as it is stored in the NCBI databases (<ProteinID>). The last column contains the total counts resulting from the RNA mapping (<Total count>).
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| Submission date |
Feb 06, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Marc Stevens |
| E-mail(s) |
marc.stevens@uzh.ch
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| Organization name |
University of Zurich
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| Department |
Vetsuisse
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| Lab |
Institute for Food Safety and Hygiene
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| Street address |
Winterthurerstrasse 272
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| City |
Zürich |
| ZIP/Postal code |
8057 |
| Country |
Switzerland |
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| Platform ID |
GPL19749 |
| Series (1) |
| GSE65716 |
Transcriptome response of Salmonella enterica subsp. enterica serovar Typhimurium N-15 and Bifidobacterium thermophilum RBL67 in co-culture |
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| Relations |
| BioSample |
SAMN03332442 |
| SRA |
SRX868586 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1603454_RBL67_co_2_Processed.xlsx |
115.7 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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