Promethion platform

Three leech species—namely, H. nipponia, H. manillensis, and W.
pigra—were obtained from the bank of Changjiang River, and their intestinal
tracts were removed and washed with saline solution. The genomic DNA was collected using
the DNeasy Blood & Tissue Kit (Qiagen, Wroclaw, Poland). The DNA quality was assessed,
a long-read library was constructed (insert size, 20 kb), and a Nanopore PromethION
platform was used to perform long-read sequencing. Hi-C was performed using the following
protocol: the leech tissues were fixed in 1% formaldehyde solution. Nuclear chromatin was
obtained from the fixed tissue and digested using HindIII (New England
Biolabs [NEB], Ipswich, MA, USA). The overhangs were blunted with bio-14-dCTP (Invitrogen,
Carlsbad, CA, USA) and Klenow enzyme (NEB). After dilution and religation using T4 DNA
ligase (NEB), the genomic DNA was extracted and sheared to 350–500 bp with a Bioruptor
(Diagenode, Seraing, Belgium). Then, the biotin-labeled DNA fragments were enriched with
streptavidin beads (Invitrogen).

Total genomic DNA of the tigecycline-resistant isolate was extracted by Puregene Yeast/Bact Kit B (Qiagen, Maryland, US), and was sequenced by using Hiseq 4000 system (Illumina, San Diego, US) and PromethION platform (Nanopore, Oxford, UK). Hybrid assembly was performed by using Unicycler version 0.4.8 [28 (link)]. Antibiotic resistance genes were identified by ResFinder 3.2 [29 (link)] and CARD (https://card.mcmaster.ca/) with identity >80% and coverage >60%. Plasmid replicon typing was performed using PlasmidFinder v2.1 (https://cge.cbs.dtu.dk/services/PlasmidFinder/) with at least 95% identity and 60% coverage. Synteny analysis was performed using Easyfig [30 (link)]. Fragments >5 kb that were absent in at least one genome were detected by BLAST and were defined as genomic islands (GEIs) in this study as previously described [31 ]. Phylogenetic analysis with amino-acid sequences of Tet(X)s was performed by using the maximum likelihood method with default parameters by using Mega X Version 10.0.5 [32 (link)]. The amino acid sequences of Tet(X)s were submitted to ESPript 3 server [33 (link)] to perform the alignment and predict the secondary structure elements.

Genomic DNA from 794 CRKP isolates was extracted using Gentra Puregene Yeast/Bact. Kit (Qiagen, San Francisco/Bay Area, CA, USA). The genomes were sequenced using an Illumina Novaseq 6000 system (Illumina, San Diego, United States) with 2 × 150-bp paired-end libraries. Raw reads were trimmed using Trimmomatic v0.33^{42 (link)} and then assembled using SPAdes v3.12.0^{43 (link)}. We performed long-read sequencing on representative isolates using a Nanopore PromethION platform (Nanopore, Oxford, UK) following a 10-Kbp library protocol. A hybrid assembly was generated by using Unicycler 0.4.0^{44 (link)} with short and long reads. QUAST v4.6.0^{45 (link)} was used to generate assembly statistics. Species were determined using FastANI v1.33 (https://github.com/ParBLiSS/FastANI), with a cut-off of 95%. The assemblies were annotated using Prokka v1.14.6^{46 (link)}.

A single colony of E. coli isolates was selected and cultured in LB medium at 37 °C. Genomic DNA was extracted using a Gentra Puregene Yeat/Bact.Kit (Qiagen, Chatsworth, CA, USA). The harvested DNA was detected by the agarose gel electrophoresis and quantified by a Qubit^® 2.0 Fluorometer (Thermo Scientific, Waltham, MA, USA). According to the manufacturer’s instructions, the harvested DNA was subjected to WGS on the Illumina NovaSeq 6000 system (Illumina, San Diego, CA, USA), which generated 150-bp paired-end reads from a library with an average insert size of 350 bp. The E. coli EC2641 was further sequenced by a Nanopore PromethION platform (Nanopore, Oxford, UK) following a 10-Kbp library protocol.

Selected seedlings were exposed to a simulated frost treatment (−2°C) for 0 h, 2 h, and 9 h. Three samples were performed for each cultivar, and each sample contained 12 plants. Leaves at the third and fourth nodes from the tip were collected and stored at −80°C for further analysis.
RNA samples were prepared using an RNA simple Total RNA Kit (DP411, TIANGEN), RNA integrality was tested by agarose gel (LabChip GX, Agient2100), and the RNA concentration was detected using a NanoDrop 2000 (Thermo Fisher Scientific).
Library preparation was performed according to the standard protocol provided by ONT. A mass of 1 μg total RNA was prepared for cDNA libraries using the cDNA-PCR Sequencing Kit (SQK-LSK110+EXP-PCB096) protocol provided by ONT. The template-switching activity of reverse transcriptases enriches full-length cDNAs and adds defined PCR adapters directly to both ends of the first-strand cDNA, followed by cDNA PCR for 14 cycles with LongAmp Tag (NEB). The PCR products were then subjected to ONT adaptor ligation using T4 DNA ligase (NEB). Agencourt XP beads were used for DNA purification according to the ONT protocol. The final cDNA libraries were added to the PromethION Flow Cell (R9 Version, FLO-PRO002, Nanopore) and run on the PromethION platform at Biomarker Technology Company (Beijing, China).