title: ONT Amplicon-Seq of cocktail bacteria culture in brain heart infusion broth
design: Multiplex RT-PCR amplicon sequencing was conducted using 4-hour cocktail culture of E. coli O157:H7, S. enteritidis, and L. monocytogenes in BHI or LJE. RNA extraction and DNA digestion were performed and verified using the NEB Monarch Total RNA Miniprep Kit including DNase I. Prior to RT-PCR amplicon sequencing, an end repair/A tailing step (NEBNext End Repair/dA-tailing Module) was carried out for the RT-PCR products, followed by a ligation step using NEB Ultra II ligation master mix. Oxford Nanopore Ligation Sequencing Kit SQK-LSK108 and Library Loading Bead Kit EXP-LLB001 were used for the library preparation of RT-PCR amplicon.