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Sample GSM5960268

Query DataSets for GSM5960268

Status

Public on Jul 12, 2022

Title

MOPS-treated S. aureus, 20 minutes post-treatment, replicate 1 [M-20-1_S101]

Sample type

SRA

Source name

S. aureus UAMS-1

Organism

Staphylococcus aureus

Characteristics

strain: UAMS-1
treatment: 50 mM MOPS pH 7.2 (final concentration); control
time point: 20 minutes post-treatment

Treatment protocol

At the time of treatment, selected cultures were treated with 1 M MOPS or 5 mg ASL/ml; the final concentration of MOPS in all cultures was 50 mM. To determine the effects of treatments at different times post-treatment, cell pellets were harvested at 20 min. and 2 hours post-treatment.

Growth protocol

Overnight cultures were diluted to OD600=0.025 in TSB (3% w/v) with a flask: volume ratio of 10:1 and grown for two hours at 37°C and 250 rpm prior to treatment.

Extracted molecule

total RNA

Extraction protocol

Cells were washed with nuclease-free Tris-EDTA buffer, then pelleted and stored at -80°C for less than 30 days prior to RNA extraction. Cells were lysed with a FastPrep-24 (MP Biomedicals, Solon, OH) and total RNA was extracted with QIAGEN RNeasy prep kits (QIAGEN, Germantown, MD), and genomic DNA was degraded with the Ambion Turbo DNA-free kit (Austin, TX). RNA samples were submitted to the University of Florida ICBR Gene Expression core to obtain RNA integrity number (RIN) values using an Agilent Bioanalyzer 2100 (Agilent Technologies, Inc, Santa Clara, CA).
Extracted RNA was submitted for sequencing to Novogene (Sacramento, CA), where RNA sample quality was also assessed by BioAnalyzer High Sensitivity RNA Assay (Agilent Technologies Inc., Santa Clara, CA) and quantified by Qubit 2.0 RNA HS assay (ThermoFisher, Waltham, MA). Ribosomal RNA depletion was performed with Ribo-Zero Plus rRNA Removal Kit (Illumina Inc., San Diego, CA). Samples were randomly primed and fragmented based on the manufacturer’s recommendation. The first cDNA strand was synthesized with the Protoscript II Reverse Transcriptase with an extension period of approximately 40 minutes at 42⁰C. All remaining steps for library construction were executed according to the NEBNext® Ultra™ II Non-Directional RNA Library Prep Kit for Illumina® (New England BioLabs Inc., Ipswich, MA). Final library quantities were assessed by Qubit 2.0 (ThermoFisher, Waltham, MA) and quality was assessed by TapeStation D1000 ScreenTape (Agilent Technologies Inc., Santa Clara, CA). Average final library size was about 375 bp with an insert size of about 200 bp. Illumina® 8-nt dual-indices were used.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

MOPS-20 min vs. Lignin-20 min.txt

Data processing

Equimolar pooling of libraries was performed based on QC values and sequenced on an Illumina® Novaseq with a read length configuration of 150 paired end (PE) for 13.5M PE reads per sample (6.75M in each direction).
Raw data files (.fastq) were imported into CLC Genomics Workbench 21 (QIAGEN, Hilden, Germany). All subsequent data processing was performed using CLC genomics workbench, unless otherwise specified.
Trim-read function was run using default parameters to remove adapter sequences.
sequences were subjected to an in silico rRNA depletion by mapping them to a sequence file containing all of S. aureus rRNA genes.
The unmapped reads (those that did not align to rRNA genes) were then mapped to a gene track generated from a MRSA252 genome (NCBI accession# NC_002952) sequence file that contains small RNA (sRNA) annotations, RPKM values calculated, and subjected to quantile normalization
Supplementary files format and content: tab-delimited text files include normalized RPKM values for each sample

Submission date

Mar 17, 2022

Last update date

Jul 12, 2022

Contact name

Kelly Rice

E-mail(s)

[email protected]

Organization name

University of Florida

Street address

1355 Museum Drive