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| Status |
Public on Nov 21, 2009 |
| Title |
AU1054 CF1, Replicate 1 |
| Sample type |
SRA |
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| Source name |
AU1054 CF-grown cells
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| Organism |
Burkholderia cenocepacia |
| Characteristics |
condition: cystic fibrosis lung environment
strain: AU1054
growth phase: mid-log phase, OD600~1.0
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| Treatment protocol |
Cell growth was monitored in triplicate 30 mL cultures of each strain under each condition. When the O.D.600 reached approximately 1.0 or 0.8 for CF and SE conditions, respectively, cells were centrifuged at 6000 rpm at 4°C and cell pellets were resuspended in 3 mL phosphate-buffered saline. One mL of culture was subjected to RNA purification by RNeasy Midi Kit (Qiagen, Valencia, CA) and eluted in 500 uL of RNase-free water. Samples were treated with DNaseI to remove any residual DNA and purified by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. Ten micrograms from each total RNA sample was subjected to further purification by enriching for mRNA via the MICROBExpress kit (Ambion) according to the manufacturer’s instructions. Samples were resuspended in 15 uL RNase-free water. cDNA was generated according to instructions given in Invitrogen’s Superscript Double-Stranded cDNA Synthesis Kit. Briefly, each mRNA sample was mixed with 100 pmol random hexamers (Integrated DNA Technologies, Coralville, IA), incubated at 70°C for 10 min, chilled on ice, mixed with 5 uL First-Strand Reaction Buffer (Invitrogen), 2.5 uL 0.1 M DTT, 1.25 uL 10 mM RNase-free dNTP mix, 3 uL Superscript III reverse transcriptase, and incubated at 50°C for 2 h. To generate the second strand, the following Invitrogen reagents were added: 86 uL RNase-free water, 30 uL Second-Strand Reaction Buffer, 3 uL 10 mM RNase-free dNTP mix, 10 U E. coli DNA Ligase, 40 U E. coli DNA Polymerase, 2 U E. coli RNase H, and incubated at 16°C for 2 h. Ten units of T4 DNA Polymerase (Invitrogen) were added the reactions were incubated for an additional 5 min at 16°C. Reactions were stopped by adding 10 uL 0.5 M RNase-free EDTA and purified by phenol-chloroform extraction and ethanol precipitation. Residual RNA was removed by treating with RNaseH and RNaseA followed by phenol-chloroform extraction and ethanol precipitation.
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| Growth protocol |
All cells were grown on a rotary shaker in 125 mL flasks containing 30 mL of medium. The cultures grown on synthetic CF sputum medium (6) at 37°C were inoculated from cells grown on LB agar plates. The inoculum for the soil extract medium (SE) was grown overnight in 1/10th tryptic soy broth at 22°C, centrifuged at 6000 rpm at room temperature, and the pellets were resuspended in 2 mL spent supernatant. One ml was diluted into 30 mL of moderate soil medium (15), which contains 10% soil extract (prepared by autoclaving 400 g sieved maize rhizosphere soil per liter of water for 15 min, centrifuging, and filtering to remove particulates) and supplemented with 3 mM glucose (equal to the CF medium). The soil cultures were incubated at 22°C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Libraries were prepared for sequencing according to the manufacturer's instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
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| Data processing |
Sequencing reads were mapped to the genome using BlastN with a threshold e-value of 0.0001 and using the "-F F" parameter. Reads mapped to rRNA and reads not mapped under the mentioned parameters were removed from further analysis. The number of reads overlapping each gene based on GenBank annotation was recorded. Reads from replicate samples were pooled, and the number of reads per gene was normalized according to the total number of reads in each library and the gene size. The resulting number is the gene expression index (GEI, expressed as normalized reads per kilobase). Reproducibility was assessed by plotting replicate GEI values against each other.
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| Submission date |
Nov 20, 2009 |
| Last update date |
Jun 11, 2013 |
| Contact name |
Deborah R. Yoder-Himes |
| Organization name |
Harvard Medical School
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| Department |
Microbiology and Molecular Genetics
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| Lab |
Lory Lab
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| Street address |
77 Louis Pasteur Ave, 1038 NRB
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL9702 |
| Series (1) |
| GSE19115 |
Mapping the Burkholderia cenocepacia niche response via high-throughput sequencing |
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| Relations |
| BioSample |
SAMN02196568 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM473848_071011_HWI-EAS127_0003_FC14657.4.fasta.txt.gz |
42.2 Mb |
(ftp)(http) |
TXT |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
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