|
| Status |
Public on Jun 19, 2016 |
| Title |
con_RNAA |
| Sample type |
SRA |
| |
|
| Source name |
Bacterium
|
| Organism |
Escherichia coli |
| Characteristics |
strain: O157:H7 NCTC 12900
treatment: None
|
| Treatment protocol |
In order to generate the VBNC state in E. coli O157:H7, 20 mL of the exponential-phase cell suspensions in a 50 mL sterile glass tube was treated by HPCD at 5 MPa and 25℃ for 40 min.
|
| Growth protocol |
The strain was stocked in tryptic soy broth (TSB) with 25% glycerol at -80℃, and was activated by streaking onto TSA plate and incubating at 37℃ for 24 h. And then, a single colony was picked and used to inoculate TSB and incubated at 37℃ for 12 h with shaking at 200 rpm. This overnight culture was transferred to TSB at a dilution of 1:100 and grew to the exponential phase (OD550=0.93).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the VBNC cells and the exponential-phase cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The RNA samples were treated with DNase Ⅰ (Invitrogen) to remove residual genomic DNA.
10 μg of total RNA sample was subjected to purification for discarding rRNA via the MICROBExpress kit (Ambion) according to the manufacturer’s protocol. Following purification, the RNA was interrupted into short fragments using divalent cations under elevated temperature and the short fragments were used for the cDNA synthesis using a SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s instructions. These cDNA fragments were purified with a QIAquick PCR purification kit (Qiagen), and then went through end reparation, adding poly(A) and ligation of sequencing adaptors. These products were purified with agarose gel electrophoresis and fragments in the size of 200-250 bp were selected for PCR amplification to construct the cDNA library.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Base calling performed using Illumina GA pipeline 1.6.
The clean reads obtained were aligned to the genome sequence of E. coli O157:H7 EDL933 using SOAP2
The raw reads were filtered for removing dirty raw reads which contain adapters, unknown or low quality bases.
All those uniquely mapped reads were used to calculate the gene expression level by the RPKM method, which is able to eliminate the influence of different gene length and sequencing discrepancy on the calculation of gene expression and thus the calculated gene expression level can be directly used for comparing the difference of gene expression among samples.
Genome_build: ASM666v1; NC_002655.2
Supplementary_files_format_and_content: Microsoft excel spreadsheet files include RPKM values for each gene of sample
|
| |
|
| Submission date |
Oct 16, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Feng Zhao |
| E-mail(s) |
zhaofeng1985@gmail.com
|
| Organization name |
China Agricultural University
|
| Department |
College of Food Science and Nutritional Engineering
|
| Street address |
No. 17 Qinghua East Road, Haidian District
|
| City |
Beijing |
| ZIP/Postal code |
100083 |
| Country |
China |
| |
|
| Platform ID |
GPL14548 |
| Series (1) |
| GSE62394 |
Transcriptomic analysis of VBNC Escherichia coli O157:H7 induced by HPCD by high-throughput Illumina sequencing technology |
|
| Relations |
| BioSample |
SAMN03112800 |
| SRA |
SRX733764 |
