title: ATCC_B1 with RA-CH-2[Tet(X)] gDNA isolate No.2
design: The genomic DNA (gDNA) of Riemerella anatipestifer RA-CH-2, which contains the tetracycline resistance gene tet(X) and erythromycin resistance gene ere(D),served as the donor, with ATCC_B (contains blaRATA-2 gene) as the recipient strain. Natural transformants were obtained through selection with sub-inhibitory concentrations of tetracycline. Sample testing included concentration, sample integrity and purity. Concentration was detected by fluorometer or Microplate Reader (e.g. Qubit Fluorometer, Invitrogen). Sample integrity and purity were detected by Agarose Gel Electrophoresis (Concentration of Agarose Gel:1% Voltage:150V, Electrophoresis Time:40 min).1g genomic DNA was randomly fragmented by Covaris.The fragmented genomic DNA were selected by Agencourt AMPure XP-Medium kit to anaverage size of 200-400bp.Fragments were end repaired and then 3 adenylated. Adaptors were ligated to the ends of these 3 adenylated fragments.This process was to amplify fragments with adaptors from previous step. PCR products were purified by the Agencourt AMPure XP-Medium kit.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.Library was qualified by QC.The qualified libraries were sequenced by BGISEQ-500: ssCir DNA molecule formed a DNA nanoball (DNB) containing more than 300 copies through rolling-cycle replication. The DNBs were loaded into the patterned nanoarray by using high density DNA nanochip technology. Finally, pair-end 100 bp reads were obtained by combinatorial Probe-Anchor Synthesis (cPAS).