|
| Status |
Public on Oct 01, 2012 |
| Title |
UCD 153 1h 3 |
| Sample type |
RNA |
| |
|
| Source name |
UCD 153 1h 3
|
| Organism |
Nostoc punctiforme PCC 73102 |
| Characteristics |
genotype/variation: wild type
|
| Treatment protocol |
For nitrogen step-down culutres supplemented with 2.5 mM NH4Cl at a chlorophyl a concentration of 2-3 ug/ml were harvested at 1000xg for 5 min, washed 3x with AA/4 and resuspended in AA/4.
|
| Growth protocol |
Nostoc punctiforme strains were cultured in Allan and Arnon medium diluted four fould (AA/4), as previously described (Campbell et al. 2007. Global gene expression patterns of Nostoc punctiforme in steady-state dinitrogen-grown heterocyst containing cultures and at single time points during the differentiation of akinetes and hormogonia. J. Bacteriol 189:5247-5256) with the exception that cultures were supplemented with 30mM fructose.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extractions were performed as previously described with a bead beater and phenol (Campbell et al. 2007. Global gene expression patterns of Nostoc punctiforme in steady-state dinitrogen-grown heterocyst containing cultures and at single time points during the differentiation of akinetes and hormogonia. J. Bacteriol 189:5247-5256).
|
| Label |
Cy3
|
| Label protocol |
Ten micrograms of total RNA were used for cDNA synthesis using SuperScript Double Stranded cDNA Synthesis Kit (Invitrogen). 0.5 ug of cDNA was labled with Cy3 dye using the Nimblegen One-color DNA labeling kit.
|
| |
|
| Hybridization protocol |
Two micrograms of labled cDNA were prepared for hybridization using the Nimblegen hybridization kit and sample tracking controls and hybridized to a Nimblegen 12x135k slide fitted with an HX12 mixer at 42C on a Nimblegen Hybridization System 4 hybridizer for 16-20h.
|
| Scan protocol |
Following hybridization slides were washed with Nimblegen wash buffer and scanned using a GenePix 4000B scanner using GenePix Pro-6.0 software (Molecular Devices).
|
| Data processing |
Scanned images (.tif) were imported into NimbleScan software (NimbleGen) where the image containing all 12 arrays on each slide was separated into 12 individual image files. An automatic alignment was performed for each individual array and X, Y, and Signal files (.xys) were generated. Normalization of .xys files was performed using the R package oligo (Carvalho et al, 2007. Exploration, normalization, and genotype calls of high-density oligonucleotide SNP array data. Biostatistics 8:485-489) and the R package oneChannelGUI (Sanges et al, 2007. oneChannelGUI: A graphical interface to Bioconductor tools, designed for life scientists who are not familiar with R language. Bioinformatics 23:2406-3408) was used to compute a linear model fit and contrasts to generate top tables containing the change in expression between experimental and reference data points as M-values (Log2[experimental]-log2[reference]) along with statistical information such as p-, adjusted p- and B-values. All data and statistical analyses were computed from three independent biological replicates. Contrasts were computed for the patN-deletion strain using the wild-type strain as a reference at each time point (i.e. the ∆patN strain t = 3 h - wild type t = 3 h) (∆patN -wild type contrast) as well as for each time point using t = 0 of the same strain as a reference (i.e. the ∆patN strain t = 3 h – the ∆patN strain t = 0h) (∆patN or wild type time course contrast). Differential expression of genes with B-values > 0 was considered statistically significant. Heat maps were generated using Genesis (Sturn et al, 2002. Genesis: Cluster analysis of microarray data. Bioinformatics 18:207-208) with the M-values from top tables for each time point.
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| |
|
| Submission date |
Aug 21, 2012 |
| Last update date |
Oct 01, 2012 |
| Contact name |
Douglas D Risser |
| E-mail(s) |
[email protected]
|
| Organization name |
UC Davis
|
| Department |
Microbiology
|
| Lab |
Meeks
|
| Street address |
One Shields Ave
|
| City |
Davis |
| State/province |
California |
| ZIP/Postal code |
95616 |
| Country |
USA |
| |
|
| Platform ID |
GPL15966 |
| Series (1) |
| GSE40250 |
Comparative transcriptomics of wild-type (UCD 153) and patN-deletion strains (UCD 524) of Nostoc punctiforme ATCC 29133 |
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