|
| Status |
Public on Oct 03, 2022 |
| Title |
8325-4phi13kan, - Mitomycin C, Rep 1 [1-8325-4DF-phi-13Kana-delta-rep_TSS] |
| Sample type |
SRA |
| |
|
| Source name |
8325
|
| Organism |
Staphylococcus aureus |
| Characteristics |
strain: 8325
variant: single-lysogen
prophage: phi13kana-Δrep
treatment: control
|
| Treatment protocol |
Samples were treated by induction with mitomycin C (300 ng/ml) or without treatment of mitomycin C as a control for 1 h. Each strain and condition was performed in biological triplicates
|
| Growth protocol |
Samples of 8325-4phi13kana and MW2cphi13kana were grown to OD600 = 0.7 (late exponential growth phase) at 37°C,200 rpm in TSB.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial pellets were resuspended in TRIzol (Thermo Fisher Scientific) and mechanically lysed using zirconia/silica beads in a high speed homogenizer. RNA was further purified using the ExpressArt® RNA ready Add-on Kit for TRIzol extraction (AmpTec) with the following modifications. After loading the sample on an RNAready column, RNA was washed additionally with inhibitor removal buffer (5 M guanidine-HCl, 20 mM Tris-HCl pH 6.6, 37 % (v/v) EtOH). DNase digest of the sample was directly performed on the column.
Library preparation on rRNA depleted RNA samples was performed as follows: first Illumina TruSeq sequencing adapter (CTGAAGCT) was ligated to RNAs containing a 5´monophosphate end (resulting from processing events and thereby represent PSS) followed by treatment with TEX (Terminator Exonuclease, Lucigen) to remove unligated 5´P-ends. Next, RNA 5´Polyphosphatase (5´PP, Lucigen) was used to convert triphosphate groups at 5´-RNA ends to monophosphate 5´-RNA ends. Formed monophosphate ends were then tagged by ligation of a second Illumina TruSeq sequencing adapter (TAATGCGC) (representing TSS).
After fragmentation, an oligonucleotide adapter was ligated to the 3´ end of RNA fragments and cDNA synthesis performed using M-MLV reverse transcriptase. cDNA was PCR amplified within 16 cycles using high fidelity DNA polymerase. cDNA was purified using Agencourt AMPure XP Kit (Beckman Coulter Genomics)
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
5´P Íllumina TruSeq sequencing tag TAATGCGC to enrich TSS after TEX-treatment
|
| Data processing |
library strategy: tagRNA-seq
CLC Genomics Workbench 20.0.3
Sequence reads were trimmed for adaptor sequence list using CLC genomic benchwork (parameter- Quality trim = yes / Quality limit: 0.05 / PolyA-tail <10)
Trimmed sequence reads were mapped 8325-4phi13Kana or MW2cphi13Kana genomic sequence using CLC genomic benchwork (mismath cost: 2; insertion cost: 3; deletion cost: 3; length fraction: 0.8; similarity fraction: 0.8)
Read count extraction and normalization (of unassigned (trimmed)-files) resulting in RPKM values were performed using CLC genomic benchwork
Transcriptional start site prediction was performed with TSS.fastq.gz-files as the enriched libraries and PSS.fastq.gz-files as control normal libraries using TSSpredator. A detailed description of TSSpredator parameters, TSS classes and output files can be taken from the UserManual available at: http://it.inf.uni-tuebingen.de/?page_id=190.
Assembly: NCBI ID: S. aureus NCTC 8325: NC_007795.1; S. aureus MW2: NC_003923.1, both strains were manually phagecured and phi13 prophage sequence inserted manually into hlb gene
Supplementary files format and content: txt format files (GE): RPKM values calculated from unassigned (trimmed)-data
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| |
|
| Submission date |
Sep 30, 2022 |
| Last update date |
Oct 03, 2022 |
| Contact name |
Christiane Wolz |
| E-mail(s) |
christiane.wolz@uni-tuebingen.de
|
| Organization name |
University Tübingen
|
| Street address |
Elfriede-Aulhorn-Strasse 6
|
| City |
Tübingen |
| ZIP/Postal code |
72076 |
| Country |
Germany |
| |
|
| Platform ID |
GPL24034 |
| Series (1) |
| GSE214523 |
Influence of Staphylococcus aureus strain background on Sa3 phage life cycle switches |
|
| Relations |
| BioSample |
SAMN31104848 |
| SRA |
SRX17761080 |
