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| Status |
Public on Sep 22, 2016 |
| Title |
MG1655_anaero |
| Sample type |
SRA |
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| Source name |
MG1655_anaero
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| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain: MG1655
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| Biomaterial provider |
ATCC 700926
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| Growth protocol |
Escherichia coli strains E.coli C (DSMZ 4860), E. coli Crooks (DSMZ 1576), E. coli DH5α (DSMZ 6897) E. coli W (DSMZ 1116), E. coli W3110 (DSMZ 5911) were obtained from DSMZ-German Collection of Microorganism and Cell Cultures; E. coli BL21 (DE3) was purchased as competent cells from Agilent (Agilent Technologies Inc., USA), E. coli K-12 MG1655 (ATCC 700926). All strains were cultured in M9 minimal medium (1) containing Na2HPO4 x 7H2O (6.8 g), KH2PO4 (3 g), NaCl (0.5 g), NH4Cl (1 g), MgSO4 (2 mmol), CaCl2 (0.1 mmol), trace elements, Wolf’s vitamin solution (2) and glucose (2 g L-1). Anoxic M9 minimal media with glucose was obtained by flushing solution with oxygen free nitrogen (95%). Overnight cultures from single colonies of each of seven E. coli strains were diluted to a starting optical density (OD600) of 0.01. Cultures were grown in 250 ml flasks or 300 ml oxygen-free sealed bottles containing 50 ml glucose-M9 minimal media in a shaking incubator at 37°C and 250 rpm.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested from aerobic and anaerobic cultures of seven E. coli strains grown to an OD600 of 0.6 (exponential phase). Cultures were divided into 10 ml aliquots and were immediately mixed with 0.2 volumes of ice-cold STOP solution (95% ethanol, 5% phenol (pH 4.7)). After 20 min incubation on ice, samples were spun down for 10 min at 4˚C and 7000 x g in a centrifuge. Pellets from one aliquot were gently resuspended in RNAProtect (QIAGEN, Germany) to further stabilize the RNA. Remaining samples were mixed with RNAlater (QIAGEN, Germany) and placed at -80˚C for archival storage. Total RNA was extracted using RNeasy Mini kit (QIAGEN, Germany) and on column DNase treatment following the manufacturers’ instructions. The 23S and 16S rRNAs were removed by subtractive hybridization using the MICROBExpress kit (Ambion, USA) with modifications. Compared with the standard protocol, 50% more capture oligonucleotides and magnetic beads were used. 5S rRNAs (120 nt in length) were removed during the total RNA extraction on column. Specifically, ribosomal depletion on total RNA isolated from the E. coli BL21 (DE3) was performed using RiboZero (Gram Negative Bacteria) kit (Epicenter, USA). RNA samples were stored at -80°C.
The sequencing libraries were constructed using the TruSeq RNA Sample Preparation kit (Illumina Inc., USA). Each library was prepared with RNA isolated from seven E. coli cultures grown in triplicate to an exponential phase under aerobic and anaerobic conditions. RT-PCR was performed with SuperScript® II One-step RT-PCR reagents (Invitrogen, USA). The libraries were sequenced using the Illumina HiSeq2000 platform with a paired-end protocol and read lengths of 50 nt. The final concentration of DNA and RNA was measured using a Qubit 2.0 Fluorometer (Invitrogen, USA). The integrity of total RNA, DNA contamination, removal of rRNAs and cDNA library validation were assessed with Agilent 2100 Bioanalyzer (Agilent Technologies, USA).
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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 |
Gene expression data (3 biological replicates per strain) were analyzed in the statistical software program R (www.r-project.org ) using the EdgeR package (3). Data were normalized using the CQN package, which accounts for both gene length and GC content effects (4). Differentially expressed genes were determined by comparing expression values under anaerobic and aerobic conditions. Those genes with adjusted P values less than 0.01 (i.e., false discovery rate less than 1%) were identified as significantly differentially expressed genes. Finally, gene annotations were automatically made using the biomaRt package (5) together with the annotation files available at the Ensembl database (www.ensembl.org) and gene set enrichment analysis (GSEA) was performed using the piano package (6). All R packages used in this study are available in Bioconductor (www.bioconductor.org).
Supplementary_files_format_and_content: tab-delimited text files include gene expresion values for each Sample
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| Submission date |
Feb 29, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Jonathan Monk |
| E-mail(s) |
jonathan.m.monk@gmail.com
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| Phone |
9716457785
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| Organization name |
UCSD
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| Street address |
9226A Regents Rd
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| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL15010 |
| Series (1) |
| GSE78756 |
Quantifying variation within the bacterial species E. coli |
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| Relations |
| BioSample |
SAMN04521252 |
| SRA |
SRX1605106 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2075731_MG1655_anaero.csv.gz |
52.7 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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