Phenol-Based RNA Isolation Protocol

RNA isolation was performed by the phenol as follows extraction based on a previously described protocol^{56 (link)}: overnight 10 mL cultures were centrifuged at 4 °C and 4816×g for 15 min and immediately used for RNA isolation. After removal of the medium, cells were suspended in 0.5 mL of ice-cold TE buffer (pH 8.0) and kept on ice. All samples were divided into two 2 mL screw-capped tubes containing 0.5 g of zirconium beads, 30 μL of 10% SDS, 30 μL of 3 M sodium acetate (pH 5.2), and 500 μL of Roti-Phenol (pH 4.5−5.0, Carl Roth GmbH). Cells were disrupted using a FastPrep-24 apparatus (MP Biomedicals) at 5500 r.p.m. for 45 s and centrifuged at 4 °C and 9400×g for 5 min. 400 μL of the water phase from each tube was transferred to a new tube, to which 400 μL of chloroform−isoamyl alcohol (Carl Roth GmbH) was added, after which samples were centrifuged at 4 °C and 18,400×g for 3 min. 300 μL of the aqueous phase was transferred to a new tube and mixed with 300 μL of the lysis buffer from the high pure RNA isolation kit (Roche). Subsequently, the rest of the procedure from this kit was performed according to the manufacturer’s protocol, except for the DNase incubation step, which was performed for 45 min. The concentration and integrity of cDNA was determined using Nanodrop-1000 Integrity and concentration of the isolated RNA was checked on a NanoDrop 1000.