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| Status |
Public on Feb 13, 2018 |
| Title |
WT Rep3 |
| Sample type |
SRA |
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| Source name |
exponentially grown cells
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| Organism |
Staphylococcus aureus |
| Characteristics |
strain: USA300_P23
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| Treatment protocol |
1 ug/ml of streptozotocin and floxuridine was treated for 3 hr
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| Growth protocol |
Cultures of strains USA300_P23, USA300_P23_streptozotocin treated, and USA300_P23_floxuridine treated in TSB media at 37ºC. Starter cultures were used to inoculate 3 mL cultures of TSB broth in 15 mL test tubes.
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| Extracted molecule |
total RNA |
| Extraction protocol |
To isolate RNA for RNA-sequencing, overnight culture in TSB was diluted 100 times in a fresh TSB and incubated in a shaking incubator at 37°C for 2 h, followed by treatment of streptozotocin and floxuridine for 3 h. After immediate stabilization of RNA in all samples by RNAprotect Bacteria Reagent (Qiagen), cells were collected by centrifugation, suspended in 50 mM of Tris HCl (pH 8) and treated with lysostaphin (50 µg/mL final concentration) at 37°C for 10 min. The resulting protoplasts were collected by centrifugation and RNA isolation was done using RNeasy Mini Kit (Qiagen), including on-column treatment with DNase I (Qiagen), according to the manufacturer’s recommendations. The amount and purity of all RNA samples isolated were assessed by NanoDrop spectroscopy (Thermo Fisher) and sent to the Center for Genomics and Bioinformatics at Indiana University. Sequencing libraries were constructed using the ScriptSeq Complete Kit for Bacteria (Epicentre).
cDNA libraries were prepared from total RNA by the Center for Genomics and Bioinformatics at Indiana University. mRNA was enriched from two micrograms total RNA using RiboZeroTM rRNA Removal (Gram-positive bacteria) Kit (EpiCentre Inc., Madison, WI, USA). rRNA-depleted mRNA samples were purified by ethanol precipitation and quantified by fluorometry with the Qubit® RNA assay kit (Invitrogen, Carlsbad, CA, USA).Double stranded cDNA synthesis was performed following ScriptSeqTM v2 RNA-Seq Library Preparation guide (EpiCentre Inc., Madison, WI, USA) in accordance with the manufacturer’s standard protocol. Thirty nanograms of enriched mRNA was fragmented using divalent cations via incubation for 5 min at 85oC. The first strand of cDNA was synthesized by reverse transcription using random-sequence primers containing a tagging sequence at their 5’ ends. Di-tagged cDNA was synthesized by random annealing of a terminal-Tagging Oligo (TTO) to the 3’ end of the cDNA for extension of the cDNA by DNA polymerase. Di-tagged cDNA was purified using Agencourt AMPure® XP beads (Beckman Coulter, Indianapolis IN, USA) followed by PCR amplification for 15 cycles using FailsafeTM PCR enzyme and ScriptSeq Index DNA primer set (EpiCentre Inc., Madison, WI). This step generated the second strand of cDNA and completed the addition of Illumina adapter sequences incorporating a user-defined barcode. The amplified libraries were purified using Agencourt AMPure® XP beads. Quality and quantity were assessed using an Agilent DNA 1000 chip (Agilent Technologies, Inc., Santa Clara, CA, USA) and Qubit® dsDNA HS assay kit (Invitrogen, Carlsbad, California, USA), respectively. Libraries were standardized to 2 μM. Cluster generation was performed using standard Cluster kits (v3) and Illumina Cluster Station.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Bases were called using bcl2fasta v2.16.0.10
Using Trimmomatic ver. 0.33 (Bolger et al., 2014), reads were adaptor trimmed and quality filtered setting the cutoff threshold for average base quality score at 20 over a window of 3 bases. Reads shorter than 15 bases post-trimming were excluded.
Cleaned reads were mapped to the complete genome of Staphylococcus_aureus_USA300_FPR3757, complete genome (CP000255.1) and the three plasmid sequences (CP000256.1, CP000257.1 and CP000258.1) using bowtie2 ver. 2.2.6.
For each library, number of uniquely mapped reads overlapping with annotated genes are counted using htseq-counts ver. 0.5.4p1.
Genome_build: Staphylococcus_aureus_USA300_FPR3757, complete genome (CP000255.1) and the three plasmid sequences (CP000256.1, CP000257.1 and CP000258.1)
Supplementary_files_format_and_content: The counts files contain counts of uniqulely mapped reads overlapping with annotated genes. Each table has two columns - Gene ID, Count of uniquely mapped reads overlapping with that gene.
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| Submission date |
Sep 20, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Ram Podicheti |
| Organization name |
Indiana University
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| Street address |
1001 E 3rd St, JH 044
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| City |
Bloomington |
| State/province |
IN |
| ZIP/Postal code |
47405 |
| Country |
USA |
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| Platform ID |
GPL24034 |
| Series (1) |
| GSE104069 |
Changes in relative transcript amounts caused by treatment of streptozotocin and floxuridine in S.aureus USA300 |
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| Relations |
| BioSample |
SAMN07676974 |
| SRA |
SRX3202115 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2788914_P23_5.counts.txt.gz |
11.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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