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| Status |
Public on Apr 17, 2024 |
| Title |
WT::pamA biological replicate C |
| Sample type |
SRA |
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| Source name |
USA300 LAC*
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| Organism |
Staphylococcus aureus |
| Characteristics |
strain: USA300 LAC*
genotype: USA300 LAC*::pamA
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| Treatment protocol |
not treated
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| Growth protocol |
overnight cultures of separate single colonies of WT::pamA and WT::EV were diluted 1:100 in 15 mL RPMI medium and incubated at 37°C shaking 180 rpm for five hours to exponential growth (OD ~0.9).
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| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA extraction, cells were isolated with centrifugation and resuspended in 1 mL Trizol (Invitrogen, #15596026), then transferred to lysing matrix B (MP Biomedicals, #116911050) tubes for mechanical disruption at 6 m/s for 30 seconds three times. After disruption, samples were centrifuged for 10 minutes at 12,000 rpm at 4°C and the upper phase was transferred into a new RNA-free tube containing 500 µL ice cold Trizol, gently mixed, and incubated for 5 minutes at room temperature, then 200 µL chloroform was added and samples were centrifuged at 12000 rpm for 15 minutes at 4°C. The aqueous phase was mixed with 500 µL isopropanol and transferred to RNeasy column (Qiagen #74004) for washing and RNA elution per manufacturer protocol.
Extracted total RNA was visualized on the Agilent 2100 Bioanalyzer system using a Bioanalyzer Nanochip run with the Prokaryote setting. RIN scores for the total RNA measured from 9 to 10. Total RNA (500 ng per sample) was input into the Illumina stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit (96rxn, cat # 20040529). Libraries were prepared according to manufacturer’s instructions. PCR Amplification was run with 11 total cycles. Following completion of the preparation, library samples were visualized on the Agilent 4200 Tapestation System using High Sensitivity DNA Screentape. The concentration of each library was assessed by Qubit using the High Sensitivity DNA Kit. The samples were pooled equimolar and the pool was sequenced on the Illumina Novaseq 6000 system on one lane of the SP 100 cycle flow cell kit. The library pool was sequenced as paired end 50 bases.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
RU121C
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| Data processing |
Raw reads were mapped to the Staphylococcus aeureus LAC genome assembly (NCBI accession GCF_015475575) , pJC1111 and pamA using Bowtie2
featureCounts was used to generate a read count table.
Assembly: GCF_015475575 + pJC1111 + pamA
Supplementary files format and content: featureCounts.tsv contains the RNA sequencing reads.
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| Submission date |
Jan 09, 2024 |
| Last update date |
Apr 17, 2024 |
| Contact name |
Robert Ulrich |
| E-mail(s) |
Robert.Ulrich@nyulangone.org
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| Organization name |
NYU Langone Health
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| Street address |
430 East 29th Street, Room 507
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10016 |
| Country |
USA |
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| Platform ID |
GPL27158 |
| Series (1) |
| GSE252862 |
Prophage-encoded methyltransferase upregulates virulence to drive adaptation in an outbreak of community-acquired methicillin-resistant Staphylococcus aureus |
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| Relations |
| BioSample |
SAMN39326326 |
| SRA |
SRX23147533 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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