Genomic analyzer

Genomic DNAs were extracted according to standard approaches [29 ]. Genome sequencing of S. olivaceus FXJ8.012Δ1741 was performed using Illumina genomic analyzer (Beijing Genomics Institute, Shenzhen, China), resulting in a total of 8,326,611 bp of genome sequence distributed across 519 contigs (≥500 bp). The genome of S. sp. FXJ1.235 was sequenced using an Illumina Hiseq system (Novogene, Beijing, China), resulting in a total of 8,852,829 bp of genome sequence distributed across 48 contigs (≥600 bp). The sequences obtained were annotated using Glimmer 3.02 [30 (link)], GeneMarkS [31 (link)], and BlastP [14 (link)]. The DNA sequences of mycemycin gene clusters have been deposited in the GenBank database. The accession number for mym is MG837055 and the numbers for mye are MG837053 (part I: myeC, myeQ, and orf-1~orf-3) and MG837054 (part II: the other mye genes and orf-4~orf-6).

The RNA extraction, library preparation and sequencing analyses were performed by the BGI Company (Shenzhen, China). The total RNA was extracted from 8 groups using RNAiso Plus according to the manufacturer’s protocols. The quality and quantity of RNA were measured by the Agilent 2100 Bioanalyzer System (Agilent Technologies, Inc., Santa Clara, CA, USA). Each RNA sample was divided into two parts, with one used for mRNA library preparation and sequencing and the second part used for RT-qPCR validation. Oligo (dT) magnetic beads were used for the enrichment of mRNAs with a poly-A tail. Purified mRNA was fragmented into small pieces with fragment buffer at the appropriate temperature. Then, first-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. The purified double-stranded cDNA was repaired, A-tails were added to the ends and the products were purified again after PCR amplification to finally obtain a single-stranded circular DNA library. The quality of cDNA was checked using the Agilent 2100 Bioanalyzer System. mRNA sequencing was performed using the Illumina genomic analyzer.

The RNA extraction, library preparation and sequencing analyses were performed by the BGI Company (Shenzhen, China). Briefly, 1 µg of each pooled total RNA was used to create small RNA libraries. RNA fragments (18–30 nt) were isolated from total RNA by polyacrylamide gel electrophoresis (PAGE). Small RNA libraries were prepared in accordance with the manufacturer’s instructions (Illumina, San Diego, CA, USA). Library creation uses a sequential addition of first a 3′ adapter sequence followed by a 5′ adapter sequence. A complimentary DNA (cDNA) copy was then synthesized using ProtoScript reverse transcriptase (New England Biolabs, Ipswich, MA, USA) and a primer complimentary to a segment of the 3′ adapter. The cDNA was amplified using reverse transcriptase PCR (RT-PCR) for 12–15 PCR cycles to complete the libraries. The quality of cDNA was checked using the Agilent 2100 Bioanalyzer system (Agilent Technologies Inc.). Libraries were then submitted to BGI (Shenzhen, China) for small RNA sequencing (RNA-seq) using the Illumina genomic analyzer.