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| Status |
Public on Aug 09, 2016 |
| Title |
rpoE LSP |
| Sample type |
SRA |
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| Source name |
Total RNA from bacteria
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| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium str. ST4/74 |
| Characteristics |
growth conditions: Growth in Lennox broth to OD600 2.0 + 6hr
strain: ST4/74 delta-rpoE
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| Growth protocol |
Wild-type and mutant cells were grown overnight in 4 ml Lennox (L-) broth (10 g/l tryptone, 5 g/l yeast extract, 5 g/l NaCl) and diluted the next morning (1:1,000) in 250 ml flasks in 25 ml L-broth and grown at 37°C and 220 rpm. A New Brunswick Innova 3400 water bath was used to grow all agitated bacterial cultures prior to RNA isolation. Bacteria were grown to mid exponential growth phase (MEP; OD600 0.3), intermediate exponential growth phase (IEP; OD600 0.8), early stationary phase (ESP, OD600 2.0) and late stationary phase (LSP, OD600 2.0 + 6h) and RNA was isolated. For growth in phosphate carbon nitrogen (PCN) minimal medium, wild-type and mutant cells were grown over night in 4 ml L-broth and the next morning 1 ml culture was washed three times with, and then diluted 1:500 in 250 ml flasks in 25 ml of, SPI2-inducing PCN (pH5.8, 0.4 mM Pi). RNA was isolated at OD600 of 0.3.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol (Invitrogen) and digested with DNase I.
cDNA libraries were constructed by Vertis Biotechnologie AG (Freising) using an in-house protocol. Total RNA (DNase I digested) was fragmented by ultrasound (4 pulses of 30s at 4°C). 3′ ends were subjected to poly(A)-tailing using poly(A) polymerase, pyrophosphate groups were removed from the 5′ terminus using tobacco acid pyrophosphatase (TAP), and an RNA adapter was ligated to the 5′ end of the RNA. First-strand synthesis was performed using an oligo(dT) adapter and M-MLV-RNaseH-reverse transcriptase, followed by PCR amplification of cDNA using high-fidelity DNA polymerase to ∼20–30 ng/μL. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Reads were de-multiplexed and poly-A tails were removed.
Sequence reads were then mapped against the S. Typhimurium 4/74 reference genome using Segemehl with accuracy set to 100%.
To increase mapping quality, unmapped reads were sequentially truncated by one nucleotide from the end of the read and were mapped again. This process was iterated until a minimal read length of 20 nucleotides or until a read mapped into a single location on the chromosome.
Genome_build: RefSeq Genome Accession: PRJNA56087 ID: 56087. Salmonella enterica subsp. enterica serovar Typhimurium str. 4/74. Richardson EJ et al., "Genome sequences of Salmonella enterica serovar typhimurium, Choleraesuis, Dublin, and Gallinarum strains of well- defined virulence in food-producing animals.", J Bacteriol, 2011 Apr 8;193(12):3162-3.
Supplementary_files_format_and_content: Wig files (.wig) are provided showing uniquely mapped sequence reads. Ready to load into Jbrowse. The file consists of two columns showing nucleotide position on the chromosome and number of reads mapped at each location. "Plus" and "Minus" refer to the two strands of the Salmonella chromosome
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| Submission date |
Mar 16, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Jay Hinton |
| E-mail(s) |
jay.hinton@liv.ac.uk
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| Organization name |
University of Liverpool
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| Department |
Institute of Integrative Biology
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| Street address |
Crown Street
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| City |
Liverpool |
| ZIP/Postal code |
L69 7ZB |
| Country |
United Kingdom |
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| Platform ID |
GPL17562 |
| Series (1) |
| GSE79314 |
Transcriptional profile of Salmonella enterica serovar Typhimurium 4/74 wild-type and mutants using strand-specific RNA-seq |
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| Relations |
| BioSample |
SAMN04559925 |
| SRA |
SRX1639005 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2091455_V2_rpoE.minus.wig.gz |
4.9 Mb |
(ftp)(http) |
WIG |
| GSM2091455_V2_rpoE.plus.wig.gz |
4.8 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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