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| Status |
Public on Oct 23, 2020 |
| Title |
Py_Fu_A_minus_TEX |
| Sample type |
SRA |
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| Source name |
Py_Fu_minus_TEX
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| Organism |
Sulfurospirillum multivorans |
| Characteristics |
strain: DSMZ 12446T
electron acceptor: Fumarate
treatment: mock treatment without the enzyme (-TEX)
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| Treatment protocol |
RNA was isolated from S. multivorans cells, harvested after four transfers on 40 mM pyruvate and 10 mM nominal PCE containing medium at an OD578 ≈ 0.26 and after 64 transfers on pyruvate and fumarate (40 mM each) at an OD578 ≈ 0.43. The cell suspension (PCE-cultivated cells: 30 ml, fumarate-cultivated cells: 18 ml) was mixed by inversion with 1/6 amount of 95% v/v ethanol / 5% v/v Roti-Aqua-phenol followed by 10 min centrifugation at 1700 x g at 4 °C. The cells were snap-frozen in liquid nitrogen and stored at -80 °C.
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| Growth protocol |
S. multivorans (DSMZ 12446T) was cultivated under anaerobic conditions at 28°C in a defined mineral medium (Scholz-Muramatsu et al., 1995) without vitamin B12 (cyanocobalamin) and yeast extract. Pyruvate (40 mM) was used as electron donor and fumarate (40 mM) or PCE as electron acceptor. PCE was added to the medium (10 mM nominal concentration) from a hexadecane stock solution (0.5 M). Pre-cultures were grown in rubber-stoppered 200 ml glass serum bottles and the main cultures in rubber-stoppered 2 l glass bottles. The ratio of aqueous to gas phase was always 1:1. In order to generate S. multivorans cells with down-regulated pceA gene expression (John et al., 2009), the organism was cultivated for 60 transfers on pyruvate (40 mM) and fumarate (40 mM). The resulting culture was used as inoculum for all pre-cultures. The bacterial growth was photometrically monitored by measuring the optical density at 578 nm.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen cell pellets were thawed on ice and resuspended in 600 µl lysis solution containing 0.5 mg/ml lysozyme in TE buffer, pH 8.0, and 60 µl 10% SDS. The cells were lysed by incubating the samples for 1-2 min at 64 °C. After the incubation, 1 M NaOAc, pH 5.2 (66 µl) was added and the sample was mixed by inversion. Total RNA was extracted using the hot-phenol method by adding 750 µl phenol. The solution was mixed by inversion and incubated for 6 min at 64 °C. Afterwards, the samples were mixed 6-10 times by inversion and cooled on ice. After centrifugation for 15 min at 14000 x g at 4 °C the aqueous layer was transferred and the chloroform extraction was performed in a 2 ml Phase Lock Gel tube (Eppendorf, Hamburg, Germany). 750 µl chloroform was added and mixed by inversion. After centrifugation for 12 min at 14000 x g at 15 °C, the aqueous layer was used for the ethanol precipitation. To the RNA containing sample, 0.1 volume 3 M NaOAc, pH 5.2 and two volumes of 96% ethanol (-20 °C) were added. The sample incubated for 2 h at -20 °C. Ethanol was discarded after centrifugation for 20 min at 14000 x g and 4 °C. The RNA was washed once with 200 µl -20 °C 70% ethanol. Ethanol was removed and the RNA was subjected to library preparation.
The libraries were generated by Vertis Biotechnologie AG (Munich, Germany). In brief, the RNA samples were poly(A)-tailed using poly(A) polymerase. Terminator exonuclease treatment (+TEX) and mock treatment without the enzyme (-TEX) were carried out after poly(A)-tailing. In this way, corresponding cDNA pairs were generated. Then, the 5'PPP structures were removed using tobacco acid pyrophosphatase (TAP). Afterwards, an RNA adapter was ligated to the 5’-monophosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and were analyzed by capillary electrophoresis. A library-specific barcode for multiplex sequencing was included as part of a 3'-sequencing adapter.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Demultiplexing
Fastq quality trimming using FastX version 0.0.13 and a cut-off value of 20
Fastq to fasta conversion using FastX
poly(A) trimming (READemption 0.3.5, Förstner et al., 2014)
Size filtering: discarding reads shorter than 12 nt (READemption 0.3.5, Förstner et al., 2014)
Read mapping using segemehl version 0.2.0 (Hoffmann et al., 2009) with an accuracy cut-off of 95% (READemption 0.3.5, Förstner et al., 2014)
Coverage calculation and normalization (READemption 0.3.5, Förstner et al., 2014)
Genome_build: CP007201.1
Supplementary_files_format_and_content: wiggle
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| Submission date |
Oct 18, 2019 |
| Last update date |
Oct 23, 2020 |
| Contact name |
Thorsten Bischler |
| E-mail(s) |
thorsten.bischler@uni-wuerzburg.de
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| Organization name |
University of Wuerzburg
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| Department |
Core Unit SysMed
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| Street address |
Josef-Schneider-Straße 2 / D15
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| City |
Würzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
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| Platform ID |
GPL27633 |
| Series (1) |
| GSE139083 |
Tetrachloroethene respiration in Sulfurospirillum species is regulated by a two-component system as unraveled by comparative genomics, transcriptomics, and regulator binding studies |
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| Relations |
| BioSample |
SAMN13056203 |
| SRA |
SRX7018967 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4130711_Py_Fu_A_minus_TEX_forward.wig.gz |
7.8 Mb |
(ftp)(http) |
WIG |
| GSM4130711_Py_Fu_A_minus_TEX_reverse.wig.gz |
7.6 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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