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SRX9401570: GSM4873501: E. coli 042 wt (aggR3UTRFRT control); Escherichia coli 042; RNA-Seq
1 ILLUMINA (NextSeq 500) run: 20.1M spots, 1.5G bases, 520.1Mb downloads

Submitted by: NCBI (GEO)
Study: Transcriptomic study of E. coli 042 with a variant of aggR gene with a FRT sequence inserted in its 3´UTR by RNA-seq
show Abstracthide Abstract
A common genomic feature of most EAEC strains is the presence of a virulence plasmid termed pAA. Plasmid-encoded virulence determinants are, among others, a transcriptional activator termed AggR, a member of the AraC-XylS family of transcription factors. We have previously determined the direct correlation between (p)ppGpp, expression of AggR and biofilm development in strain EAEC 042 (https://doi.org/10.3389/fmicb.2018.00717). In this work we characterize a novel variant of the aggR gene. We modified its 3´UTR by insertion of a FRT sequence, which have generated a series of different phenotypes. We used RNA-seq to compare the transcriptome of the wt strain and its aggR3UTRFRT variant grown at 37ºC in LB medium. Overall design: E. coli 042 and derivative aggR3UTRFRT mRNA profiles were generated by Illumina RNA-seq. RNA extraction, DNase treatment, evaluation of RNA quality and cDNA libraries for Illumina sequencing were performed by Vertis Biotechnologie AG, Freising-Weihenstephan, Germany. Bacterial cells were grown until O.D.600 of 2.0 in LB medium at 37ºC with 200rpm of agitation. Three replicates were grown from each strain, 1 ml of each replicate was collected and bacterial pellets were maintained at -80ºC. Bacterial pellets were sent to Vertis Biotechnologie AG, Freising-Weihenstephan, Germany in dry ice. E. coli 042 wt was used as genome reference (NC_017626 for the chromosome and NC_017627 for the pAA plasmid) for analysis.
Sample: E. coli 042 wt (aggR3UTRFRT control)
SAMN16596285 • SRS7619504 • All experiments • All runs
Library:
Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Total RNA was isolated from the cell pellets using the RNAsnap protocol developed by Stead et al. 2012. The total RNA preparations were examined by capillary electrophoresis. From the total RNA samples, ribosomal RNA molecules were depleted using the in-house developed depletion probes. The ribodepleted RNAs were first fragmented using ultrasound (2 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' ends of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. For Illumina NextSeq sequencing, the cDNAs were pooled in approximately equimolar amounts. The cDNA pool was fractionated in the size range of 200 – 500 bp using a differential clean-up with the Agencourt AMPure kit. An aliquot of the size fractionated cDNA pool was analyzed by capillary electrophoresis. The cDNAs have a size range of 200 - 500 bp. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The cDNA pool was paired-end sequenced on an Illumina NextSeq 500 system using 75 bp read length.
Experiment attributes:
GEO Accession: GSM4873501
Links:
Runs: 1 run, 20.1M spots, 1.5G bases, 520.1Mb
Run# of Spots# of BasesSizePublished
SRR1293790320,076,5171.5G520.1Mb2021-10-31

ID:
12272557

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