Transcriptomics of Cyanobacterium Synechocystis

Synechocystis 6803 cultures were harvested by rapid filtration on hydrophilic polyethersulfone filters (Pall Supor 800 Filter, 0.8 µm). The filter covered with cells was immediately immersed in 1 ml of PGTX solution^{26 (link)} and frozen in liquid nitrogen. Total RNA was extracted and analysed by gel electrophoresis and Northern blotting as described.^{27 (link)} For each condition, total RNA from two independent cultures was pooled for the following analyses. For sequence analysis, cDNA libraries were constructed (vertis Biotechnologie AG, Germany) and analysed on an Illumina sequencer as previously described.^{23 (link)} The dRNA-seq protocol^{22 (link)} distinguishes treated and untreated libraries. For the treated libraries, total RNA was fragmented with ultrasound (four pulses of 30 s at 4°C) and RNA species that carry a 5′ mono-phosphate were degraded using Terminator™ 5′ phosphate-dependent exonuclease (TEX, Epicentre). The exonuclease-resistant mRNA (mainly primary transcripts with 5′-PPP) was poly(A)-tailed using poly(A) polymerase. Then, 5′-PPP RNA was cleaved enzymatically using tobacco acid pyrophosphatase (TAP), ligated to an RNA linker^{23 (link)} and first-strand cDNA synthesis initiated using an oligo(dT)-adapter primer and M-MLV reverse transcriptase. The second-strand cDNA synthesis was primed with a biotinylated antisense 5′-Solexa primer, after which cDNA fragments were bound to streptavidin beads. Bead-bound cDNA was blunted and 3′ ligated to a Solexa adapter. The cDNA fragments were amplified by 10–12 cycles of PCR. The resulting cDNA samples were double-stranded with a size of ∼150–700 bp. For the untreated libraries, we pooled total RNA from samples representing all 10 growth conditions and depleted rRNA using the MICROBExpress kit (Ambion). Except for the TEX treatment, the libraries were handled as described for the treated libraries.