Extraction of the whole genome and total RNA
Single colony was picked up from XM solid medium and inoculated into TSB liquid medium (Sigma, Germany) in flasks. 5 mL cell samples were collected after the shaking incubation at 34 °C for 48~72 h and were used for genome extraction. 400 to 600 μL TSB culture was centrifuged at 12000 rpm for 3 min for pellet collection. Pellets were then washed by 600 μL 0.05 M N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid sodium salt (TES) buffer (pH=8.0) twice and resuspended in 700 μL TES buffer. 50 mg/mL of lysozyme and 4 mg/mL RNase were sequentially added to a final concentration of 5 mg/mL or 40 μg/mL, respectively. Pellets with enzymes were incubated at 37 °C for at least 1 h. The tubes should be gently inverted 4 to 5 times during incubation until the liquid became sticky. 10% sodium dodecyl sulfate was added to a final concentration of 1~2%. 5 μL of 20 mg/mL protease K was also needed for protein denaturation. The mixture was then incubated at 37 °C for 30 min, during which the tubes were vigorously inverted 2 to 3 times until the liquid was transparent. To precipitate the protein, 100 μL of 5 M NaCl and 50 μL of 10% CTAB preheated to 65 °C were added in tubes. The mixture was then incubated at 65 °C for 1 h. The tubes were inverted each 15 min. To extract DNA from the liquid, equivalent volume of phenol:chloroform:isoamyl alcohol (25:24:1) saturated with 10 mM Tris, pH 8.0, 1 mM EDTA was used and then vibrated in vortex for 15 s. The mixture was centrifuged at 12000 rpm for 10 min. Clear supernatant was transferred into new tubes and extraction by phenol:chloroform:isoamyl alcohol (25:24:1) was repeated 3 times. To precipitate DNA, 3 M NaAc was added to a final concentration of 0.3 M. 4 °C isopropanol was added before DNA samples were kept in -20 °C overnight. The mixture was then centrifuged at 12000 rpm for 1 min at 4 °C and the supernatant was discarded. 2 mL cold ethanol was used to wash samples and the supernatant was discarded after 12000 rpm centrifugation for 10 min. 1 mL cold 70% ethanol was then added and supernatant was also discarded. At last, 30 to 50 μL TE buffer preheated to 55 °C was added in tubes to dissolve genome. Concentration of genome was measured by NanoDrop spectro-photometer. Genome sequencing was performed on Illumina Hiseq2000 platform (BGI, China).
For RNA isolation, two replicates from independent cultures in modified minimal liquid cultures were used. Cells were harvested at two time points, i.e. 10 h in the early exponential phase and 50 h at the onset point of erythromycin biosynthesis, for total RNA extraction. 10 mL culture was centrifuged at 4000×g at 4 °C for 10 min. The broth supernatant was discarded and the cells were resuspended in 2 mL RNAlaterTM solution (Invitrogen, USA). Total RNA was extracted with an RNeasy Plus Mini kit (Qiagen, Germany) using glass beads to mechanically disrupting cells with a FastPrep (30 s ×4; M.P. Biomedical, USA). DNase treatment by RNase-Free DNase Set (Qiagen, Germany) aided to digest DNA in samples. The RNA integrality was analyzed by 1% agarose gel electrophoresis and Bioanalyzer (Agilent, USA). The RNA quality was also determined by Bioanalyzer (Agilent, USA). 1 μg total RNA was reverse transcribed using a PrimeScriptTM RT Reagent Kit with gDNA Eraser (Takara, Japan) for RT-qPCR. RNA sequencing was accomplished using BGIseq 500 next-gen sequencer or Illumina Hiseq4000 sequencer, which was commercially provided by BGI, China or Shanghai Majorbio Bio-pharm Technology Co.,Ltd, respectively. The original image data obtained from sequencers was transferred into sequence data via base calling, which is defined as raw data or raw reads and saved as FASTQ file. Datasets consisted of at least 30 M pair-ended reads per sample with a 100- or 150-bp read length. After sequencing, the raw reads were filtered includes removing adaptor sequences, contamination and low-quality reads from raw reads. Especially for genome raw reads, reads with a large amount of duplications or N base were also removed.