Halobacterium NRC-1 General transcription factor perturbation

Halobacterium cells, specified general transcription factor knock-out strains or plasmid expressed 6-His tagged general transcription factor strains, were grown to mid-log phase. RNA extractions were performed using the Stratagene Absolutely RNA Miniprep Kit and RNA quality checked with the Agilent Bioanalyzer and with Oligo Microarrays were fabricated at the Institute for Systems Biology Microarray Facility. The arrays contain 4 spots per unique 70-mer oligonucleotides for each of 2400 non-redundant genes in Halobacterium NRC-1 Labeling, hybridization and washing have been previously described (Baliga et al. 2002) with 10 μg of RNA from the sample and reference. RNA from the final time point of a replicate experiment was used as the reference. . Bias in dye incorporation was accounted for by reversing the labeling dyes (dye-flip). Raw data was processed and converted into log10 ratios with lambda (λ) values determined by a maximum likelihood method. Baliga, N. S., Pan, M., Goo, Y. A., Yi, E. C., Goodlett, D. R., Dimitrov, K., Shannon, P., Aebersold, R., Ng, W. V. & Hood, L. (2002) Proc Natl Acad Sci U S A 99:14913-84. Keywords: genetic perturbation each knockout or perturbed expression strain was analyzed at a single point during growth. This sample was analyzed on duplicate microarrays as a dye flip (2 microarrays per sample).