Sample 2
Nostoc sp. PCC 7120 = FACHB-418
Protocols: Anabaena 7120 and derivatives were cultured in BG11 medium in the light, on a rotary shaker. Anabaena filaments were washed 3 times with BG11 (without nitrate) before induction of heterocyst formation in the same medium. Cells for extraction of RNA were cultured with air bubbling before and after nitrogen stepdown. Algal cells were harvested by centrifugation at 4991 g for 5 min, flash frozen with liquid nitrogen, and stored at -80 °C until use. The gene hetZ (alr0099) was knocked out by insertional inactivation and the gene hetP (alr2818) was knocked out by replacing hetP with cassette C.CE2 Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Genomic DNA was removed by digest with DNase I. Strand-specific RNA-Seq libraries were prepared using Illumina® Small RNA Sample Preparation Kit according to the manufacturer's protocol.