Halobacterium NRC-1 Diurnal experiment Light 2

The wild type strain of Halobacterium salinarium strain NRC-1 was used for the diel growth experiments. For experiment, cultures were started at an OD >> 0.001. Cultures were grown anaerobically at a temperature of 38° C under light:dark cycles (12:12 hrs) for 120 hours then grown under constant light. Average light intensity was 150 μE/m2/sec. Time course sampling began 84 hours into the light:dark phase. Samples were collected every 3 hours for 72 hours. At each time point, culture was withdrawn by syringe and replaced with nitrogen gas. Aliquots of the culture were centrifuged and the cell pellet flash-frozen in a dry ice/ethanol bath after decanting the supernatant. 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. For this experiment 3 μl of ULYSIS Alexa Fluor 594 and 6 μl of 660 dyes (Molecular Probes) were used for labeling. 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: environmental response 25 conditions were analyzed on duplicate arrays (50 total microarrays) as dye-flips.