ONT sequencing of Synechocystis sp. PCC 6803 Stockholm
ONT sequencing was performed for three Synechocystis WT substrains. The Stockholm strain, generously provided by Paul Hudson, is a non-motile glucose-tolerant substrain. The Uppsala strain, a glucose-tolerant, non-motile Synechocystis substrain, was kindly gifted by Pia Lindberg. The motile PCC-M, strain from Freiburg, which was kindly sent years ago by Annegret Wilde. Cryopreserved Synechocystis WT strains were streaked on 1% agar plates with BG11 and incubated in a Multitron HT Incubator (Infors, Switzerland) (30 C, 150 rpm shaking, 1 % CO2, 75 % humidity, 32 mol(photons)m-2 s-1. 20 ml BG11 in 100 ml Erlenmeyer flasks were inoculated and cultures were incubated under the same conditions and shaking at 150 rpm until an OD750 nm of ~7-8. Cells from 2 ml culture were harvested by centrifugation for 15 min at 10,000 rcf, resuspended in 1ml 0.1M EDTA (pH 8), and incubated for 3  h at 300 rpm and 24C to remove EPS. After centrifugation at 10,000 rcf for 10 min, the pellets were either washed once with 1 ml MiliQ (Freiburg strain) or directly stored at -20C (Uppsala and Stockholm strains) for at least one night.  Cells were resuspended in 400 l XS buffer (1% potassium ethylxanthogenate, 800 mM NH4OAc, 100 mM Tris-HCl pH 7.4, 20mM EDTA, pH8.0, 1% SDS) and incubated for ~2h at 65C, with three freeze-thaw cycles in liquid nitrogen. For DNA extraction, 1 volume of phenol:chloroform: isoamyl alcohol (25:24:1) was added and mixed gently by inversion. After centrifugation at 10,000 rcf and 20C for 10 min, the aqueous phase was retrieved,  treated with 5l RNAseA (4.5U in 10 mM Tris pH8.5, 15mM NaCl) and subsequently incubated at 37C and RT overnight. Phenol:chloroform:isoamylalcohol extraction were repeated four times before adding 1 volume of chloroform or chloroform:isoamylalcohol. Samples were gently inverted and centrifuged at 10,000 rcf and 20C for 10 min, and the aqueous phase was re-isolated twice with chloroform:isoamylalcohol. DNA in the aquesous phase was precipitated with 0.6 ml ice-cold EtOH and 0.2M NaCl (final concentration) at -20C for at least 1 night. DNA was pelleted by centrifugation at 10,000 rcf for 30 min, washed twice with 0.4 ml 70% EtOH, air-dried, and resuspended in 20 l EB buffer (Tris-HCl) by subsequent incubation at 50C and RT overnight.