Lysozime

RNA was extracted from RM and MM-grown cells originally harvested from 3 ml of culture. Total RNA isolation involved vortexing of the pellet with 6 ml of RNA Protect (QIAGEN) followed by centrifugation. The pellet was thereafter lysed using 280 μl of lysis buffer (10% Zwittergent (Calbiochem), 15 mg/ml Lysozime (Sigma) and 20 mg/ml Proteinase K (Roche) in TE buffer). Total RNA was purified with RNeasy mini kit (QIAGEN, Valencia, CA) combined with DNase I (QIAGEN) according to the manufacturer’s instructions. The quantity and quality of RNA were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technology, Rockland, DE) and Experion Automated Electrophoresis using the RNA StdSens Analysis Kit (Bio Rad).

Samples were manipulated in biosafety level 2 hoods with laminar flow. All samples were thawed on ice prior to DNA extraction. S samples were first incubated with four volumes of Sputasol (Oxoid, Hampshire, United Kingdom), mixed with four volumes of phosphate buffer solution and centrifuged at 16000 g for 10 min. The pellet obtained was mixed with 0.6 mL of a lysis buffer containing lysozime (final concentration 83 μg/mL; Sigma-Aldrich Corp., St. Louis, MO, United States), lysostaphin (final concentration 20 U/mL; Sigma-Aldrich) and mutanolysin (final concentration 250 U/mL; Sigma-Aldrich). OP samples were directly suspended on the same volume of the lysis buffer. The same procedure was followed with the pellet obtained from N samples after centrifugation. All samples were incubated during 2 h at 37°C (Yuan et al., 2012 (link)).
Samples were transferred to a DNA dry bead tube (MO BIO Laboratories, Carlsbad, CA, United States), and shaken at 5000 rpm for 30 s in a Precellys Minilys homogenizer (Bertin Technologies, Rockville, Washington, DC, United States). After digesting with Proteinase K for 1 h at 60°C, samples were again shaken in the homogenizer and DNA was purified according to the manufacturer’s instructions. Genomic DNAs were quantified using the Qubit Fluorometer (Life Technologies. Carlsbad, CA, United States).