Halobacterium NRC-1 oxygen, light and ark perturbation

A 50ml culture of Halobacterium NRC-1 knockout strain delta-ura3 with additional in-frame gene deletion of ark was grown to mid-log phase in a 125ml flask in rich media under varying oxygen and light conditions. Low and high light indicate growth in a painted flask versus growth in a standard clear flask under constant standard incubator illumination, respectively. Low and high oxygen indicate shaking in a stoppered flask at 100RPM agitation or shaking in a non-stoppered flask at 220RPM, respectively. Each of the four possible pairwise environemental combinations were assayed : High-oxygen/high-light, Low-oxygen/high-light, High-oxygen/low-light and Low-oxygen/low-light. 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: environmental response, genetic perturbation 4 samples were analyzed in duplicate (8 total microarrays) as dye-flips.

将50mL的盐杆菌（Halobacterium NRC-1）敲除菌株Δura3且额外带有ark框内基因缺失的培养物，于125mL摇瓶中在丰富培养基内、不同氧气与光照条件下培养至对数中期。低光照与高光照分别指在涂漆摇瓶与标准透明摇瓶中，于恒温培养箱恒定光照下的培养状态；低氧与高氧分别指在带塞摇瓶中以100RPM振荡培养，以及在敞口摇瓶中以220RPM振荡培养的状态。四种两两组合的环境条件均完成检测：高氧/高光照、低氧/高光照、高氧/低光照及低氧/低光照。

RNA提取采用Stratagene Absolutely RNA迷你柱式提取试剂盒，RNA质量通过Agilent Bioanalyzer生物分析仪进行质控，寡核苷酸微阵列由系统生物学研究所微阵列中心制备。该微阵列针对盐杆菌NRC-1的2400个非冗余基因，每个独特的70聚体寡核苷酸设置4个重复点样位点。标记、杂交与洗涤流程已在既往研究中详述（Baliga等，2002），实验中使用10μg样品RNA与参照RNA。以重复实验终时间点的RNA作为参照样本。通过翻转标记染料（染料翻转，dye-flip）来校正染料掺入偏差。原始数据经处理后转换为log₁₀比值，λ值通过最大似然法计算得出。

参考文献：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) 美国国家科学院院刊，99:14913-84。关键词：环境响应、遗传扰动。

4个样品均进行了重复检测（共计8张微阵列），所有重复实验均采用染料翻转策略。