Illumina HiSeq 2500 sequencing
transcriptome analysis
The harvested cells were washed with polysome buffer (20 mM Tris-HCl, pH 7.5; 140 mM NaCl and 5 mM MgCl2), and then resuspended with 500 ��L lysis buffer (0.3 M sodium acetate, pH 5.2; 10 mM ethylenediaminetetraacetic acid and 1% Triton X-100). The resuspended cells were frozen with liquid nitrogen and ground using a mortar and pestle. The ground mycelium was thawed and centrifuged at 4��C for 10 min at 16,000 �� g. The supernatant was collected and stored at ?80��C. Following the preparation of lysates from four growth phases as biological duplicates, the lysates were mixed with a solution of phenol:chloroform:isoamyl alcohol (25:24:1, v/v), and the mixtures were separated by centrifugation. Any DNA in the extracted RNA samples were removed by treatment with 2 ��L DNase I (NEB), 5 ��L 10 �� DNase I buffer and 1 ��L SUPERase-In RNase Inhibitor (Thermo Scientific, Waltham, MA, USA). Finally, the DNase I-treated RNA samples were purified using phenol:chloroform:isoamyl alcohol (25:24:1, v/v) and ethanol precipitation. To eliminate rRNAs in the recovered RNA samples, Ribo-Zero rRNA Removal Kit for Bacteria (Epicentre, Madison, WI, USA) was used according to the manufacturer��s instructions. The quality of rRNA-depleted RNA samples was checked using 2% agarose gel electrophoresis. The suitable RNA samples were then used to construct RNA sequencing libraries using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA). The size distributions of final libraries were checked using the Agilent 2200 TapeStation System (Agilent, Santa Clara, CA, USA). The constructed libraries were sequenced on the HiSeq 2500 platform using either 100-bp (S. lividans, S. avermitilis, S. clavuligerus, and S. venezuelae) or 50-bp (S. tsukubaensis) single-end read recipe.