GSM9035199: L. lactis subps. cremoris MG1363_2xMS2-sLLM1042+_rep1; Lactococcus cremoris subsp. cremoris MG1363; OTHER
25 ml of cells were harvested by centrifugation at 5,000 × g for 10 min at 4°C, resuspended in 2 ml of 1× PBS supplemented with 1 mM DTT and 0.4 mM PMSF, and homogenized twice for 40 s at 5 m/s using MP Biomedicals homogenizer. Lysates were clarified by centrifugation at 15,000 × g for 15 min at 4°C. 25 μl of Glutathione Sepharose 4B resin (Fisher Scientific) per sample was pelleted at 500 × g for 5 min and washed 3 times with 1× PBS containing 1 mM DTT and 0.4 mM PMSF. 10 μg (25 μl) of purified GST-2xMCP protein was added and incubated for 10 min at 4°C with rotation. The resin was washed twice with 0.5 ml of the same buffer. Cleared lysate was applied to the resin, followed by 4 washes with 0.5 ml of the same buffer. RNA was eluted in 1× PBS containing 1 mM DTT and 10 mM reduced glutathione. An equal volume of Roti-Aqua-PCI solution (Carl Roth) was added, mixed, and centrifuged at 13,000 × g for 5 min at 4°C. RNA was precipitated by adding 3 volumes of 99.9% ethanol, followed by centrifugation at 13,000 × g for 40 min at 4°C. Pellets were washed twice with 70% ethanol and centrifuged at 13,000 × g for 10 min at 4°C. Libraries were prepared by Novogene following DNase treatment and rRNA depletion, according to their standard prokaryotic directional mRNA library preparation protocol.