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| Status |
Public on Oct 08, 2022 |
| Title |
ER00573, 3 hours, Replicate 2 [MRSA-17] |
| Sample type |
SRA |
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| Source name |
ER00573_3 hours
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| Organism |
Staphylococcus aureus |
| Characteristics |
strain: ER00573
genotype: WT sarZ
time: 3 hours
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| Growth protocol |
Each strain was cultured for 3h (logarithmic growth) and 5h (stationary phase) in 5mL tryptic soy broth (TSB), spun down, and resuspended in 1mL RNA STAT-60TM (Amsbio).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were bead beated using FastPrep and spun down. Upper phase was collected and 200µL chloroform was added. 0.5mL isopropanol was added to aqueous phase to precipitate RNA. RNA was washed with 70% ethanol, air dried, and resuspended in RNase free water. 2500ng of RNA was DNase treated (TURBO DNA-freeTM kit, Invitrogen Ambion).
For each sample, 250ng of RNA was first depleted of bacterial rRNAs using enzymatic ribodepletion with the Illumina Ribo-Zero Plus rRNA Depletion Kit (Illumina, Catalog no. 20037135) according to the manufacturers protocol. RNA-Seq libraries were then prepared using the Illumina TruSeq Stranded Total RNA Library Prep kit using the depleted rRNA material as input and following the manufacturer’s protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw reads were first trimmed by removing Illumina adapter sequences from 3’ ends using cutadapt, and by removing 3′ read sequences if more than 20 bases with Q≥20 were present. Paired-end reads were then mapped to the FPR3757 (NC_007793) reference genome using Bowtie2, and htseq-count was used to produce strand-specific transcript count summaries. Raw fragment (i.e., paired-end read) counts were then combined into a numeric matrix, with genes in rows and samples in columns.
Differential gene expression analysis was performed with the limma R package in Bioconductor. Normalization factors were computed on the data matrix using the weighted trimmed mean of M-values (TMM) method, followed by voom 65 mean-variance transformation in preparation for Limma linear modeling. Only genes with expression levels ≥1 FPKM (fragments per kb per million reads) in at least 50% of samples, and a length ≥200 bp, were retained for further analysis. Filtered data were fitted to a design matrix containing all sample groups, and pairwise comparisons were performed between the groups of interest. Finally, eBayes adjusted P-values were corrected for multiple testing using the Holm method and used to select genes with significant expression differences (q ≤ 0.05).
Genome_build: FPR3757 (NC_007793)
Supplementary_files_format_and_content: Tab-delimited text file with normalized log (counts per million)
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| Submission date |
Oct 08, 2021 |
| Last update date |
Oct 10, 2022 |
| Contact name |
Harm van Bakel |
| E-mail(s) |
[email protected]
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| Organization name |
Mount Sinai School of Medicine
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| Department |
Genetics and Genomic Sciences
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| Lab |
Bakel Lab
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| Street address |
One Gustave L. Levy Place, Box 1498
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL27158 |
| Series (1) |
| GSE185544 |
Evolution of hypervirulence during bloodstream adaptation of CA-MRSA strain USA300 |
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| Relations |
| BioSample |
SAMN22148570 |
| SRA |
SRX12528209 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5617982_MRSA-17.genes.sens.count.txt.gz |
12.5 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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