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| Status |
Public on Aug 19, 2019 |
| Title |
scuR++ RNAseq S2 |
| Sample type |
SRA |
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| Source name |
scuR++
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| Organism |
Streptococcus salivarius |
| Characteristics |
strain/background: HSISS4
genotype/variation: scuR++
medium: CDM+glucose
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| Growth protocol |
Streptococcus salivarius and derivatives were grown at 37°C in chemically defined medium supplemented with glucose (CDMG) without shaking.
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| Extracted molecule |
total RNA |
| Extraction protocol |
S. salivarius WT, ∆scuR, ∆sarF, scuR-ST++ or sarF-ST++ strains were pre-cultured overnight in CDMG at 37°C. They were resuspended in 50 ml of fresh pre-warmed CDMG to a final OD600 of 0.05 and grown for approximately 2 h 30 min (OD600 = 0.3) at 37°C. Cells were harvested by centrifugation (10 min; 4,050 × g), the supernatants were discarded and the cell pellets were frozen with liquid nitrogen. Finally, RNA was extracted using the RiboPure bacteria kit (Ambion-Life Technologies) and the protocol provided by the manufacturer, with protocol changes to cell lysis and RNA precipitation. For lysis, cells were resuspended in RNAwiz buffer (Ambion-Life Technologies) supplemented with Zirconia beads and shaken for 40 sec (4 times) in a fastPrep homogenizer device (MP biomedicals). For RNA precipitation, a 1.25-ethanol volume (instead of 0.5) was added to partially purified RNAs. Total RNA was checked for quality on an RNA Nano chip (Agilent Technologies) and concentration was measured using Ribogreen assay (Life Technologies). rRNA depletion was performed on 2 µg total RNA with the Ribo-Zero rRNA removal kit for Gram-positive bacteria (Illumina) according to manufacturer’s instructions.
Total stranded mRNA libraries were prepped with the NEBNext Ultra Directional RNA Library Prep kit for Illumina (New England Biolabs). Library PCR was executed for 15 cycles. Quality of the libraries was evaluated with the use of a High sensitivity DNA chip (Agilent Technologies) and concentrations were determined through qPCR according to Illumina protocol. Both Libraries were equimolar pooled for sequencing on a NextSeq 500 high-throughput run with 76bp single reads. 2.3 pM of the library pool was loaded on the flowcell with a Phix spike-in of 5%.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
mRNAs
processed data: Table S2.xlsx
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| Data processing |
Sequenced mRNAs generated several million reads that were mapped on the WT S. salivarius chromosome and processed with both bowties V0.12.9 (http://bowtie-bio.sourceforge.net/bowtie2) and samtools V0.1.18 (http://samtools.sourceforge.net/) algorithms to yield BAM files containing the read coordinates. We imported these files into SeqMonk V0.23.0 (www.bioinformatics.babraham.ac.uk/projects/) to assess the total number of reads for each coding sequence (CDS). The dataset was exported into an excel file for further analyses. First, the dataset was standardized to CDS-mapped reads per million overall reads. Then, we estimated a ratio of CDS-mapped reads in mutants vs WT.
Genome_build: Streptococcus salivarius HSISS4: CP013216.1
Supplementary_files_format_and_content: Table_S1.xlsx, Table_S2.xlsx: Excel files reporting RNAseq were generated by SeqMonk sofware. The sheet is organized in columns as follows: column 1 : gene name; column 2 : genome unit; column 3 : probe start; column 4 : probe end; column 5 : gene function; column 6 : locus tag; column 7 : old locus tag assigned in the previous genome sequencing; column 8 : orientation of the probe in regard to the associated gene; column 9 : absolute value of reads per probe in the WT; column 10 : absolute value of reads per probe in the scuR mutant (deletion in Table S1 or overexpression in Table S2); column 11 : absolute value of reads per probe in the sarF mutant (deletion in Table S1 or overexpression in Table S2); column 12 : Ratio of Normalized abundance of reads per probe in scuR mutant (deletion in Table S1 or overexpression in Table S2) versus WT background; column 13 : Ratio of normalized abundance of reads per probe in sarF mutant (deletion in Table S1 or overexpression in Table S2) versus WT background; column 14 : log10 transformation of column 12; column 15 : log10 transformation of column 13.
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| Submission date |
Sep 28, 2018 |
| Last update date |
Aug 19, 2019 |
| Contact name |
Johann Mignolet |
| E-mail(s) |
[email protected]
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| Organization name |
UNIL
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| Department |
DMF
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| Lab |
Veening lab
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| Street address |
Biophore Quartier UNIL-SORGE
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| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platform ID |
GPL23620 |
| Series (1) |
| GSE120640 |
Molecular singularities in a pheromone sensor triumvirate desynchronize the competence-predation interplay in the human commensal Streptococcus salivarius |
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| Relations |
| BioSample |
SAMN10143727 |
| SRA |
SRX4776658 |
| 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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