Nextera xt library

Twenty-five ESBL-producing E. coli isolates were chosen for whole-genome sequencing (WGS). The isolates were confirmed to be ESBL-producers using the VITEK®2 compact system (bioMérieux, Nürtingen, Germany). DNA was isolated using the Purelink Genome DNA Mini kit (Invitrogen, Darmstadt, Germany). WGS was carried out on an Illumina MiSeq instrument (Illumina, San Diego, CA, USA) using an Illumina Nextera XT library with 2x300-bp paired-end reads. The data was assembled using SPAdes (version 3.0) (Bankevich et al., 2012 (link)). Contigs larger than 500-bp and a coverage higher than nine were ordered to E. coli MG1655 (accession number U00096.3) using MAUVE (Darling et al., 2004 (link)). The contigs were concatenated to generate pseudogenomes. Whole-genome phylogeny was determined by the software package Harvest Suite using E. coli MG1655 as reference (Treangen et al., 2014 (link)). The phylogenetic tree was drawn using MEGA5 (Hall, 2013 (link)).
The raw data of the sequenced E. coli are available at the European Nucleotide Archive (ENA), under the project number PRJEB12335.

Short-read sequencing libraries were prepared from the isolated DNA with the Nextera XT library preparation kit (Illumina Inc., San Diego, CA, USA) and subsequently sequenced using v3 chemistry on a MiSeq system (Illumina) in paired-end mode.
Four strains (Mukteswar, NCTC120, 34, BfR242) were additionally sequenced by nanopore long-read technology (ONT). For this purpose, libraries were prepared with the Ligation Sequencing Kit SQK-LSK 109 (Oxford Nanopore Technologies Ltd., Oxford, UK) together with the Barcoding Kit EXP-NBD 104 (Oxford Nanopore Technologies Ltd., Oxford, UK) and sequenced on an R9.4.1 flow cell with a MinION Mk1B sequencing device (Oxford Nanopore Technologies Ltd., Oxford, UK) for 24 h. Sequencing raw data and hybrid assemblies were deposited at the European Nucleotide Archive under project number PRJEB52165.

RNA samples were quantified using a Qubit 2.0 Fluorometer (Life Technologies) and RNA integrity was checked with a 2100 TapeStation (Agilent Technologies). RNA library preparations, sequencing reactions and initial bioinformatics analysis were conducted at GENEWIZ, LLC. (South Plainfield, NJ, USA). SMART‐Seq v4 Ultra Low Input Kit for Sequencing was used for full‐length cDNA synthesis and amplification (Clontech, Mountain View, CA, USA), and Illumina Nextera XT library was used for sequencing library preparation. Briefly, cDNA was fragmented and adaptor was added using transposase, followed by limited‐cycle PCR to enrich and add index to the cDNA fragments. The final library was assessed with a Qubit 2.0 Fluorometer and Agilent TapeStation.