|
| Status |
Public on Oct 18, 2014 |
| Title |
LB 0.4 B2 TEX neg L1 HS2 |
| Sample type |
SRA |
| |
|
| Source name |
Escherichia coli MG1655 cells
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: MG1655
medium: LB
growth phase: exponential
|
| Treatment protocol |
Samples were mixed, incubated on ice for 10 min after which cells were collected by centrifugation at 4150 rpm at 4˚C for 10 min. Cell pellets were snap frozen in an ethanol/dry ice slurry and stored at -80°C until total RNA could be extracted.
|
| Growth protocol |
To harvest total RNA samples, overnight cultures of wild type MG1655 grown in LB at 37˚C were diluted back 1:500 in either fresh LB or M63 minimal glucose medium and allowed to grow until the cultures reached an OD600 of ~ 0.4 and 2.0 for cultures grown in LB and an OD600 of ~0.4 for cultures grown in M63. For samples grown to OD600 of ~0.4 a total volume of 25 ml of cells were harvested and combined with 5 ml of stop solution (95% Ethanol, 5% acid phenol). For samples grown to OD600 of 2.0 a total volume of 5 ml of cells were harvested and combined with 1 ml of stop solution.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Hot phenol (Sharma et al. 2010, Bloomberg 1990, EMBO J)
The libraries were generated by Vertis Biotechnologie AG (Munich, Germany). The samples were poly(A)-tailed by using poly(A) polymerase. The 5'-PPP were removed using tobacco acid pyrophosphatase (TAP) followed by the ligation of the RNA adapter to the 5'-monophosphate of the RNA. First-strand cDNA synthesis was performed with an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNA was PCR-amplified to reach a concentration of 20-30 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. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Demultiplexing
Fastq quality trimming using FastX and a cut-off value of 20
Fastq to fasta conversion using FastX
Size filtering: discarding reads shorter than 12 nt (TRAPL)
Read mapping using segemehl version 0.13 (TRAPL)
Coverage calculation and normalisation (TRAPL)
Genome_build: NC_000913.2
Supplementary_files_format_and_content: wiggle
|
| |
|
| Submission date |
Feb 20, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Konrad U. Förstner |
| E-mail(s) |
foerstner@zbmed.de
|
| Organization name |
ZB MED - Information Centre for Life Sciences
|
| Department |
Information Services
|
| Lab |
Förstner Lab
|
| Street address |
Gleueler Str. 60
|
| City |
Cologne |
| State/province |
North Rhine-Westphalia |
| ZIP/Postal code |
50931 |
| Country |
Germany |
| |
|
| Platform ID |
GPL15010 |
| Series (1) |
| GSE55199 |
Identification and validation of antisense RNAs in Escherichia coli |
|
| Relations |
| BioSample |
SAMN02646724 |
| SRA |
SRX474168 |
