RNA-Seq of S. clavuligerus: NL strain, 72h
Cells were harvested and washed with a polysome buffer (140 mM NaCl, 20 mM Tris-HCl pH 7.5, and 5 mM MgCl2). Cell pellets were resuspended with lysis buffer (0.3 M sodium acetate pH 5.2, 10 mM EDTA, and 1 % Triton X-100). The cell resuspension was physically lysed by grinding with mortar and pestle, after it was frozen with liquid nitrogen. The supernatant was collected by centrifugation of the cell lysate at 4  for 10 min at 16000  g. It was then used for RNA extraction, or stored at 80 . The supernatant was mixed with equal volume of phenol:chloroform:isoamyl alcohol = 25:24:1 solution. After centrifugation, RNA was extracted from aqueous layer, and it was precipitated by ethanol. RNA samples were treated with DNase I (New England Biolabs, USA) to remove any DNA contaminations. TruSeq stranded mRNA (Illumina, USA) preparation kit was used for cDNA library preparation, following the manufacturers protocol.