small RNA sequencing of S. mutans
Bacteria were cultured overnight in BHI broth, then the bacterial RNA was extracted after bacterial precipitation was treated with lysozyme for 40 min at 37. All RNA fragments that were 1830 nt in length were isolated by PAGE gel. Then the 1830 nt small RNAs were ligated to a 5-adaptor and a 3-adaptor. The adapter-ligated small RNAs were subsequently transcribed into cDNA by SuperScript II Reverse Transcriptase (Invitrogen, USA) and then several rounds of PCR amplification with PCR Primer Cocktail and PCR Mix were performed to enrich the cDNA fragments. The PCR products were purified by QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA). The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library. The library was amplified with phi29 to make DNA nanoball (DNB) which have more than 200 copies of one molecular. The DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on BGISEQ500 platform (BGI-Shenzhen, China).