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SRX7018968: GSM4130713: Py_Fu_A_plus_TEX; Sulfurospirillum multivorans; RNA-Seq
1 ILLUMINA (Illumina HiSeq 2500) run: 6.1M spots, 612.3M bases, 375.8Mb downloads

Submitted by: NCBI (GEO)
Study: Tetrachloroethene respiration in Sulfurospirillum species is regulated by a two-component system as unraveled by comparative genomics, transcriptomics, and regulator binding studies
show Abstracthide Abstract
Sulfurospirillum multivorans is one of the few bacteria, which can anaerobically respire organohalides such as tetrachloroethene. The regulation of this organohalide respiration is in most parts unknown. Sulfurospirillum multivorans was shown to downregulate the expression of organohalide respiration-specific genes slowly when no substrate is present, over the time of approximately 100 generations. To unravel the molecular details of this peculiar regulation and the involved factors, we sequenced the primary transcriptome of the organism. Overall design: Sulfurospirillum multivorans was grown anaerobically with pyruvate as energy and C-source and either tetrachloroethene or fumarate as electron acceptor; RNA was isolated from four samples each for transcriptional start site analysis – two for a library treated with and two for a library treated without terminator 5'-phosphate-dependent exonuclease.
Sample: Py_Fu_A_plus_TEX
SAMN13056202 • SRS5539962 • All experiments • All runs
Library:
Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction 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.
Experiment attributes:
GEO Accession: GSM4130713
Links:
Runs: 1 run, 6.1M spots, 612.3M bases, 375.8Mb
Run# of Spots# of BasesSizePublished
SRR103076906,124,967612.3M375.8Mb2020-10-23

ID:
9222227

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