Hiseq novaseq 6000 platform

We performed WGS on carbapenem-resistant K. pneumoniae strains with the objective of gaining further insight on the molecular epidemiology, resistome, and virulome of these strains. A total of 68 nonduplicated K. pneumoniae clinical strains were selected based on the carbapenemase gene type identified and within these, by random selection when the number of strains was above one, as previously carried out [6 (link)]. The genomic DNA were extracted for WGS from cultures grown overnight in Mueller–Hinton agar, using the NZY Tissue gDNA Isolation kit (NZYTech, Lisbon, Portugal), as per the manufacturer’s recommendations. The Sequence was done at STABVida Portugal. Sequencing libraries were prepared using the KAPA HyperPrep Library Preparation Kit (Roche, Basel, Switzerland) following the manufacturer’s recommended protocol, and sequenced using an Illumina HiSeq Novaseq 6000 platform (Illumina, San Diego, CA, USA) with paired-end reads (2 × 151 bp). The raw data quality control was performed using FASTQC v0.11.9 (Babraham Institute, Cambridge, UK) and the trimming and de novo assembly were performed using CLC Genomics Workbench 12.0.3 (QIAGEN, Aarhus, Denmark). All assemblies were carried out with automatic word size, similarity fraction of 0.95, a length fraction of 0.95, and a minimum contig size of 500 bp.

To achieve a high enough RNA concentration, 2–3 mice left ventricular (LV) tissues were pooled and homogenized as one biological replicate. Total RNA was isolated from swim-trained and control mice LVs and treated with DNase I. Polyadenylated mRNA was further enriched from total RNA using Dynabeads Oligo (dT)₂₅ (Ambion, Cat#61005) following the manufacturer’s protocol. mRNA was then fragmented into ~100 nucleotide fragments using fragmentation buffer (10 mM ZnCl₂, 10 mM Tris-HCl pH7.0). Fragmented RNA was incubated for 2 h at 4 °C with affinity purified anti-m⁶A rabbit polyclonal antibody (Synaptic Systems, Cat#202003) in m⁶A Binding buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 2 mM EDTA) supplemented with BSA (0.5 μg/μL). The mixture was then immunoprecipitated by incubation with protein A beads and eluted with elution buffer (1 × IP buffer and 6.7 mM m⁶A). After precipitation with 75% ethanol, eluted mRNA-containing fragments (IP) and untreated input control fragments were used to prepare libraries with a strand-specific library by the dUTP method. The libraries were sequenced on an Illumina Hiseq Novaseq 6000 platform and 150 bp paired-end reads were generated.