For CO2-shift experiments cells were pre-cultivated for at least 24 h in liquid medium and a continuous stream of air enriched with 5% [v/v] CO2 through the culture (defined as high CO2, HC). Prior to the shift experiments, cells in exponential growth phase were harvested by centrifugation (5 min at 3000 g, 20°C), washed, re-suspended in fresh BG11 medium (pH 7.0) and adjusted to OD750 = 0.8. After approximately 1 h continued cultivation at HC, CO2 enrichment was stopped and cells were bubbled with ambient air (0.035% CO2, defined as low CO2, LC). RNA samples were taken at HC immediately before and 24 h after shift to LC by 10 min centrifugation at 6.000 rpm and 4°C.
Growth protocol
A glucose-tolerant strain of Synechocystis sp. PCC 6803 (a kind gift of N. Murata, National Institute for Basic Biology, Okazaki, Japan) was used as the wild type (WT) reference strain in this study. The ΔcyabrB2 mutant was obtained from the group of Y. Hihara, Saitama University, Japan (Ishii and Hihara, 2008). Mutant cells were maintained in the presence of 50 µg ml-1 of kanamycin in BG11 medium (Rippka et al., 1979; J Gen Microbiol 111: 1–61) that was buffered with 20 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES)-KOH at pH 8.0. The WT was cultivated without antibiotics under otherwise identical conditions. The general growth conditions were 29°C constant temperature with continuous light set to approximately 80 μmol photons m-2 s-1 and otherwise as previously detailed (Schwarz et al., 2011; Plant Physiol 155: 1640–1655).
Extracted molecule
total RNA
Extraction protocol
Cells were lysed in 1 ml PGTX, with the following composition: 39.6% (w/v) phenol, 7% (v/v) glycerol, 7 mM 8-hydroxyquinoline, 20 mM EDTA, 97.5 mM sodium acetate, 0.8 M guanidine-thiocyanate, and 0.48 M guanidine hydrochloride (Pinto et al., 2009; BMC Mol Biol 10: 79). Phenol was extracted twice with chloroform:isoamyl alcohol (24:1) and RNA was precipitated with isopropanol at -20°C, followed by centrifugation at 4°C. The RNA pellet was washed with 70% ethanol and resuspended in RNase-free A. dest.
Label
Cy3
Label protocol
The RNA was labeled directly, without cDNA synthesis in 2 µg aliquots with the Kreatech “ULS labeling kit for Agilent gene expression arrays” with Cy3 according to the manufacturers protocol.
Hybridization protocol
The labelled RNA was fragmented and hybridized as described by the manufacturer's instructions for Agilent one color microarrays with 1.65 µg of labeled RNA
Scan protocol
Arrays were scanned on the Agilent Technologies Scanner G2505B, using Agilent Feature Extraction Software 10.7.3.1 and the protocols GE1_107_Sep09 for Cy3 labelled arrays
Data processing
Raw data were processed with the R package Limma. Median signal intensity was quantile normalized.
