Magna pure 96 dna and viral na large volume kit

For assessment of SARS-CoV-2 RNA of the lung and dLN samples, unfixed and, where possible, non-cryopreserved (i.e., native) tissue samples were used. RNA was purified from ∼50 mg of homogenized tissue obtained from all samples by using the MagNAPure 96 system and the MagNAPure 96 DNA and Viral NA Large Volume kit (Roche) according to the manufacturer’s instructions. The controls without COVID-19 were validated by Rhonda PCR rapid COVID-19 test (Spindiag) according to the manufactures protocol on ∼50 mg of homogenized tissue together with a positive control.
Quantitative real-time PCR for SARS-CoV-2 was performed on RNA extracts with RT–qPCR targeting the SARS-CoV-2 E gene. Quantification of viral RNA was performed using photometrically quantified in vitro RNA transcripts⁵⁴ . Total DNA was measured in all extracts by using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific). The RT–qPCR analysis was replicated at least once for each sample.
As a correlate of active virus replication in the tested tissues, subgenomic RNA (sgRNA) was assessed by using oligonucleotides targeting the leader transcriptional regulatory sequence and a region within the sgRNA encoding the SARS-CoV-2 E gene^{55 (link)}. All information is presented in Table 1.

Among the FQR isolates from the phenotypic testing, 42 isolates with ciprofloxacin levels ≥ 0.5 µg/ml were randomly selected for whole genome sequencing (WGS). WGS was performed by MicrobesNG (MicrobesNG, Birmingham, UK.) using Illumina HiSeq technology [22 ]. DNA for sequencing was extracted using the MagNA Pure 96 DNA and Viral NA Large Volume kit (Roche Diagnostics GmbH, Mannheim, Germany) according to manufacturer’s instructions and genomic libraries were prepared using the Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA). A 150-bp paired-end sequencing was performed using the HiSeq × 10 system (Illumina). Long and short read sequences were assembled using Unicycler (v.0.4.8.0), and genome annotation was done with Prokka (v.1.14.6). Sequences were analyzed for multisequence typing (MLST), and plasmid replicon types using MLST 1.8, ResFinder [23 (link)] and Plasmidfinder software [24 (link)].

Nucleic acids were extracted from 500 μL STGG medium and eluted in a final volume of 100 μL with the MagNA Pure 96 instrument using the MagNA Pure 96 DNA and Viral NA Large Volume kit and the Pathogen Universal protocol (Roche Diagnostics, Basel, Switzerland). Amplicon sequencing of the 16S ribosomal RNA (rRNA) gene was performed as described elsewhere [20 (link)]. Briefly, a fragment of ~ 464 bp of the V3-V4 region of the 16S rRNA gene was amplified and sequenced with the MiSeq desktop sequencer (Illumina, San Diego, USA).