|
| Status |
Public on Jul 31, 2019 |
| Title |
RHA1_LB_repB |
| Sample type |
RNA |
| |
|
| Source name |
RHA1 in LB rich conditions rep2
|
| Organism |
Rhodococcus jostii RHA1 |
| Characteristics |
genotype/variation: wild-type
|
| Growth protocol |
Triplicate cultures of Rhodococcus jostii RHA1 and the Rhodococcus jostii RHA1/pGKT2 transconjugant were grown in LB media at 30°C with continuous shaking at 180 rpm until late log/early stationary phase.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After ~48 h, cultures were centrifuged at 8,000 x g for 10 min at 4°C and total RNA was extracted using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA) and according to the manufacturer’s instructions for RNA cleanup with an added enzymatic lysis step and mechanical disruption step added before cleanup. Enzymatic lysis and mechanical disruption was performed by resuspending the cell pellet in 100 uL TE buffer and 200 uL lysozyme (20 mg/mL), transferring the resuspended pellet to a MP Biomedicals (Solon, OH) Lysing Matrix B tube and bead beating for 30s. The optional on column DNA digestion using the Qiagen RNase-Free DNase Kit was performed to remove DNA. RNA quality and concentration was determined by analysis with an Agilent 2100 bioanalyzer.
|
| Label |
Cy3
|
| Label protocol |
The RNA was used to generate cRNA using the Low Input Quick Amp Labeling Kit (Agilent, Santa Clara, California) and ethanol precipitated following the manufacturer’s directions. At each step the quality of the RNA and cRNA was analyzed using the Agilent 2100 Bioanalyzer and Total RNA Kit (Agilent) and the concentration verified using the NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA).
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| |
|
| Hybridization protocol |
Three hundred and twenty-five micrograms of the labeled DNA was used for subsequent hybridization to custom Agilent 8 x 15K microarrays using the Agilent hybridization kit and following the manufacturer's protocol . The hybridized arrays were washed using the Agilent Wash Buffer Kit.
|
| Scan protocol |
Washed arrays were scanned using the Agilent Technologies, High-Resolution Microarray Scanner (Model G2505C, Agilent Technologies, Santa Clara, CA, USA)
|
| Description |
This sample is of Rhodococcus jostii RHA1 grown under aerobic batch conditions in LB media. It is the second of three biological replicates used in this experiment, each from separate cultures
|
| Data processing |
Microarray images were analyzed using Agilent Feature Extraction software and ArrayStar v4.0.0 (DNAstar, Madison, WI) software. Expression data were log2 transformed and statistical significance was determined using a student t-test for binary transcriptome comparisons (i.e. control vs. experimental). The P-value was set at 0.05 and only those genes induced or repressed at least two-fold were considered in this paper. Differentially expressed transcripts were assigned to their functional classifications based on pathway enrichment analysis from the Biocyc Database Collection (https://biocyc.org/)
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| |
|
| Submission date |
Jan 24, 2019 |
| Last update date |
Aug 02, 2019 |
| Contact name |
Karl Indest |
| E-mail(s) |
indestk@wes.army.mil
|
| Organization name |
US Army Engineer Research and Development Center
|
| Street address |
3909 Halls Ferry Road
|
| City |
Vicksburg |
| ZIP/Postal code |
39180 |
| Country |
USA |
| |
|
| Platform ID |
GPL26089 |
| Series (2) |
| GSE125548 |
Enhanced plasmid mediated bioaugmentation of RDX contaminated matrices in column studies using donor strain Gordonia sp. KTR9 [exp1] |
| GSE125551 |
Enhanced plasmid mediated bioaugmentation of RDX contaminated matrices in column studies using donor strain Gordonia sp. KTR9 |
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