Cells from 2 ml of culture were pelleted (16k x g, 60 s) and flash frozen.
Growth protocol
The parental strain (delta-ura3) was grown from colony in standard complex media (CM; media M2), before passage into each of 10 media that vary in ion composition but not total salinity. During batch growth in the experimental media, samples for microarrays were taken at OD600 ~ 0.4-0.5, to capture mid-log phase and the range where growth rate approaches maximum. Cells were batch cultured in flasks at 37°C with constant shaking (~220 rpm). The standard complex media composition is as follows: 250 g/l NaCl, 20 g/l MgSO4, 2 g/l KCl, 3 g/l sodium citrate, 10 g/l Oxoid brand bacteriological peptone. Media were supplemented with 50mg/l uracil to compensate for the uracil deficiency caused by the delta-ura3 marker. All media were at total salinity of 4.388 M. All contain the same levels of sodium citrate and peptone. The levels of NaCl, MgSO4, and KCl varied as follows. M1: 4.28 M NaCl, 0.108 M MgSO4, 0 M KCl. M2: 4.28 M NaCl, 0.081 M MgSO4, 0.027 M KCl. M5: 4.28 M NaCl, 0 M MgSO4, 0.108 M KCl. M6: 3.42 M NaCl, 0.968 M MgSO4, 0 M KCl. M7: 3.42 M NaCl, 0.726 M MgSO4, 0.242 M KCl. M9: 3.42 M NaCl, 0.242 M MgSO4, 0.726 M KCl. M12: 2.57 M NaCl, 1.364 M MgSO4, 0.454 M KCl. M13: 2.57 M NaCl, 0.909 M MgSO4, 0.909 M KCl. M14: 2.57 M NaCl, 0.454 M MgSO4, 1.364 M KCl. M15: 2.57 M NaCl, 0 M MgSO4, 1.818 M KCl
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from the cell pellets using the mirVANA RNA kit (Ambion, Austin, TX) according to manufacturer's instructions and then treated with RNase-free DNase (Promega).
Label
Cy3,Cy5
Label protocol
RNAs were direct labeled with Alexa547 and Alexa647 dyes (Kreatech). See Baliga NS, Bjork SJ, Bonneau R, Pan M, Iloanusi C, Kottemann MC, Hood L, DiRuggiero J. Systems level insights into the stress response to UV radiation in the halophilic archaeon Halobacterium NRC-1.Genome Res. 2004 Jun;14(6):1025-35.
strain: NRC-1 genotype: wild type temperature: 37°C od: 0.471
Treatment protocol
Cells from 2 ml of culture were pelleted (16k x g, 60 s) and flash frozen.
Growth protocol
The parental strain (delta-ura3) was grown from colony in standard complex media (CM; media M2), before passage into each of 10 media that vary in ion composition but not total salinity. During batch growth in the experimental media, samples for microarrays were taken at OD600 ~ 0.4-0.5, to capture mid-log phase and the range where growth rate approaches maximum. Cells were batch cultured in flasks at 37°C with constant shaking (~220 rpm). The standard complex media composition is as follows: 250 g/l NaCl, 20 g/l MgSO4, 2 g/l KCl, 3 g/l sodium citrate, 10 g/l Oxoid brand bacteriological peptone. Media were supplemented with 50mg/l uracil to compensate for the uracil deficiency caused by the delta-ura3 marker. All media were at total salinity of 4.388 M. All contain the same levels of sodium citrate and peptone. The levels of NaCl, MgSO4, and KCl varied as follows. M1: 4.28 M NaCl, 0.108 M MgSO4, 0 M KCl. M2: 4.28 M NaCl, 0.081 M MgSO4, 0.027 M KCl. M5: 4.28 M NaCl, 0 M MgSO4, 0.108 M KCl. M6: 3.42 M NaCl, 0.968 M MgSO4, 0 M KCl. M7: 3.42 M NaCl, 0.726 M MgSO4, 0.242 M KCl. M9: 3.42 M NaCl, 0.242 M MgSO4, 0.726 M KCl. M12: 2.57 M NaCl, 1.364 M MgSO4, 0.454 M KCl. M13: 2.57 M NaCl, 0.909 M MgSO4, 0.909 M KCl. M14: 2.57 M NaCl, 0.454 M MgSO4, 1.364 M KCl. M15: 2.57 M NaCl, 0 M MgSO4, 1.818 M KCl
Extracted molecule
total RNA
Extraction protocol
Total RNA was prepared from the cell pellets using the mirVANA RNA kit (Ambion, Austin, TX) according to manufacturer's instructions and then treated with RNase-free DNase (Promega).
Label
Cy5,Cy3
Label protocol
RNAs were direct labeled with Alexa547 and Alexa647 dyes (Kreatech). See Baliga NS, Bjork SJ, Bonneau R, Pan M, Iloanusi C, Kottemann MC, Hood L, DiRuggiero J. Systems level insights into the stress response to UV radiation in the halophilic archaeon Halobacterium NRC-1.Genome Res. 2004 Jun;14(6):1025-35.
Hybridization protocol
Whole-genome tiling arrays were designed using e-Array (Agilent Technologies) for the H. salinarum NRC-1 main chromosome (NC_002607), and pNRC100 (NC_001869) and pNRC200 (NC_002608) plasmids using a 60k feature design of 60-mer strand-specific probes spaced at 24 bp. Arrays included manufacturer’s control probes and were printed by Agilent Technologies. Hybridization and washing was performed as described (Turkarslan et al 2011), with dye flip experiments performed for each sample. See http://www.chem.agilent.com/Scripts/PDS.asp?lPage=34519 for details.
Normalized log10 intensity of X_INT/Y_INT (sample/reference) averaged over dye flip between sample and reference RNA.
Data processing
Spot finding was performed using Feature Extraction (Agilent Technologies). See http://www.systemsbiology.org/Scientists_and_Research/Technology/Data_Management/Microarray_Pipeline for more details. Normalization and statistical analysis were performed as described before (Koide et al, 2009). Values are normalized log10 ratios representing test/reference. Interarray comparisons were normalized relative to a reference RNA sample generated from unperturbed H. salinarum NRC-1 batch culture in the standard complex medium to OD600 = 0.471.
