RNA-Seq of S. mutans
Bacteria were cultured overnight in BHI broth, and then used for RNA extraction after bacterial precipitation was treated with lysozyme for 40 min at 37. First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 200 copies of one molecular, DNBs were loaded into the patterned nanoarray and pair end 100 bases reads were generated on BGIseq500 platform (BGI Genomics, shenzhen, China).