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Sample GSM4957709

Query DataSets for GSM4957709

Status

Public on Dec 05, 2020

Title

S_aureus_USA300_biofilm_BHI_1

Sample type

SRA

Source name

Biofilm cells

Organism

Staphylococcus aureus

Characteristics

strain: USA300
rna harvesting time: 9h

Treatment protocol

Planktonic cells (3 biological replicates) were washed with cold PBS and kept in RNA stabilization solution (25 mM sodium citrate, 10 mM EDTA, 47% ammonium sulphate, adjusted to pH 5.2), centrifuged, the supernatant discarded, and the pellet kept at -80°C until extraction. Biofilms (3 biological replicates) were washed with cold PBS and then suspended in RNA stabilization solution by scraping the bottom of the well until enough cells were harvested. Biofilms cells were then centrifuged, the supernatant discarded, and the pellet kept at -80°C until extraction

Growth protocol

200 µl of the appropriate biofilm inducing medium (TSBg(s) or BHIg(s)) is added in wells of a 48-wells culture plate and inoculated to an OD600 of 0.005 with the bacteria suspension. Planktonic cells are grown with agitation while biofilm cells are grown without agitation until RNA harvesting time (9h for S. aureus strains and 7h for E. faecalis strains)

Extracted molecule

total RNA

Extraction protocol

Cells were suspended in 250 µl of fresh lysis buffer (45 mg/ml of lysozyme, 16 µl/ml of lysostaphin 5 mg/ml and 10 µl/ml of mutanolysin 5 U/µl in Tris-EDTA) and incubated for 1h at 37°C, while mixed by every 15 min. They were then transferred in O-Ring tubes with 25 to 50 mg of acid-washed beads and mixed with 3 volumes of TRI Reagent®, to be lysed mechanically with a FastPrep-24™ Classic (MP Biomedicals, Santa Anna, CA, USA) (speed of 4.5 m/sec, 2x 20s). Total RNA was extracted with the Direct-zol™ RNA Miniprep Plus kit (Zymo Research, Irvine, CA, USA) following the manufacturer instruction, including the DNAse I (New England Biolabs, Ipswich, MA, USA) treatment in the extraction column. RNA quantity and the integrity number (RIN) were evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). To remove ribosomal RNA (rRNA), samples were treated using a Pan-Prokaryote riboPOOL™ kit (siTOOLs Biotech Gmbh, Planegg, Germany) coupled with streptavidin magnetic beads (New England Biolabs, Ipswich, MA, USA). mRNA was eluted in 50 µl of nuclease-free water and then cleaned and concentrated using a Clean & Concentrator kit (Zymo Research, Irvine, CA, USA).
Libraries were prepared for RNA sequencing according to the instructions of the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New Englands Biolabs, Ipswich, MA, USA). RNA samples were fragmented 7 min instead to produce fragments superior to 100 pb. Instead of the adaptors provided with the kit, we used TruSeq compatible YIGA adaptors. Nucleic acid purification was performed using Mag-Bind® TotalPure NGS beads (Omega Bio-Tek Inc, Norcross, GA, USA) following the recommended ratio of the NEBNext® kit. Ligation products were subject to PCR amplification using a reverse primer containing a unique 8 bp index for each sample. Concentration of the libraries was measured using a Quant-iT™ PicoGreen™ dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA) and 1.13 ng of each sample were pool in 80 µl of nuclease-free water. Quantity and quality of the final mix were confirmed with an Agilent 2100 Bioanalyzer before submitting.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

Geneious_DEGs_S_aureus_USA300_Biofilm_vs_planktonic_in_BHI.txt

Data processing

Trimming with BBDuk plug-in for Geneious Prime software version 2020.0.3. Kmer length : 27. Discarding short read (minimum 10 pb). Trimming low quality (both end, minimum 6).
Trimmed data were then aligned with Bowtie2 plug-in for Geneious Prime software version 2020.0.3 using an “end-to-end” alignment type, a “High Sensitivity / Medium” preset and using the “do not trim option”, against the following reference chromosomes.
Expression quantification was performed with the build-in tool of Geneious Prime software version 2020.0.3, calculating RPKM, FPKM and TPM for each annotation on the reference sequence. Ambiguously mapped reads counted as partial matches.
Differentially expressed genes (DEGs) were assed with DESeq2 by combining the 3 biological replicates of each condition and comparing biofilm cultures vs planktonic cultures in either TSBg(s), BHIg(s) or both. Only the genes with an adjusted P-value ≤ 0.05 were retained for further analysis. DEGs were consider upregulated if they had a log2 fold change ≥ 2.0 and downregulated if they had a log2 fold change ≤ -2.0. For the transcriptomic study on media effect on genes expression, DEGs with a log2 fold change ≥ 2.0 are considered upregulated in BHIg(s) while DEGs with a log2 fold change ≤ 2.0 are considered upregulated in TSBg(s).
Genome_build: GenBank accession number GCA_000013425.1 (for S. aureus SH1000, a close descended of the ancestral strain NCTC 8325); GenBank accession number GCA_000017085.1 (for S. aureus USA300); GenBank accession number GCA_000742975.1 (for E. faecalis ATCC 29212) and GenBank accession number GCA_000007785.1 (for E. faecalis V583).
Supplementary_files_format_and_content: tab-delimited text files including Locus Tag, Gene name (if available), Description (if available), Minimum, Maximum, cds length, Direction, nucleotide sequence, Differential Expression Adjusted P-value, Differential Expression Log2 Ratio, Differential Expression Wald Statistic, Differential Expression Absolute Confidence and Differential Expression Base Mean.

Submission date

Dec 04, 2020

Last update date

Dec 05, 2020

Contact name

Pascale B. Beauregard

E-mail(s)

pascale.b.beauregard@usherbrooke.ca

Organization name

Université de Sherbrooke

Department

Département de Biologie