Halobacterium NRC-1 Diurnal experiment Dark 2

The wild type strain of Halobacterium salinarium strain NRC-1 was used for the diel growth experiments. Culturing from colony was done in a liquid Complete Medium (CM; at 42 ºC with shaking at 100 rpm). For experiment, cultures were started at an OD >> 0.001. Cultures were grown aerobically (shaking at 100 rpm) and a temperature of 42° C under light:dark cycles (12:12 hrs) for 72 hours then grown in complete darkness. Light intensity was 150 μE/m2/sec. Time course sampling began after the 72 hour light:dark phase. Samples were collected every 1 to 3 hours for 65 hours. 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 Absolute RNA kit and RNA quality checked with the Agilent Bioanalyzer and with PCR. 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. 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 18 conditions were assayed on duplicate arrays (36 total microarrays) as dye flips.