Illumina sequencing of Synechocystis sp. PCC 6803 Stockholm
Cells for Genomic DNA for Illumina sequencing was isolated from Synechocystis using a phenol-chloroform method adapted from Makowka et al. (2020). For this, 50 mL liquid culture was centrifuged (8,000 g, 10 min) and the cells were resuspended in 500 L sterile TE buffer (10 mM Tris and 1 mM EDTA, pH 7.4). Cells were broken by the addition of 10 L 10 % (w/v) SDS, 500 L phenol-chloroform-isoamylalcohol solution (25:24:1 (v/v/v)), 500 L glass beads (0.17-0.18 mm diameter), and by vortexing for 3 min at 4C. The lysate was then centrifuged (2 min, 10,000 xg, RT) and the upper phase, containing the nucleic acids, was transferred to a new centrifugation cup. For further purification, 500 L phenol-chloroform-isoamyl alcohol solution (25:24:1 (v/v/v), 4 C) was added, and the mixture was centrifuged (2 min, 10,000 xg, RT). The upper phase was transferred to a new cup, mixed with 500 L chloroform-isoamyl alcohol (24:1 (v/v)), and centrifuged (2 min, 10,000 xg, RT). This step was repeated twice, and the resulting supernatant was transferred to a new cup. A volume of 3 M sodium acetate (pH 5), equivalent to around 10% of the supernatant volume, was added, and the cup was mixed by inverting before adding a volume of absolute ethanol (-20 C) equivalent to around 250% of the supernatant volume. To precipitate the DNA, the samples were incubated at -20 C overnight, then centrifuged (15 min, 10,000 xg, 0 C). The supernatant was discarded, and the pellet was washed by adding 1 mL of 70 % (v/v) ethanol (-20 C), followed by centrifugation (5 min, 10,000 g, RT). After discarding the supernatant, the pellet was dried at 50C for around 5 min and dissolved in 20 L TE buffer. Optionally, the DNA concentration was measured with a spectrophotometer (DS-11 FX, Biozym, Hessisch Oldendorf, Germany). Samples were normalized to 20 ng/l, and 500 ng were sent in for short read NovaSeq 150 sequencing (Mircrobe-EZ, Genewiz, Germany)