Qiaamp dsp dna mini kit

Genomic DNA was extracted from cells using the QIAamp DSP DNA Mini Kit (61304; QIAGEN). After DNA was denatured with NaOH at RT for 10 min, 1,000, 500, 250, and 125 ng of DNA was blotted onto a nitrocellulose membrane (1228243; GVS). DNA was cross-linked twice using the Stratalinker UV Crosslinker (400071; Stratagene) under “AUTO CROSS LINK” mode. The membrane was blocked for 1 h at RT with gentle rocking in TBST buffer supplemented with 5% nonfat dry milk. Antibody incubation (1:2,000 dilution) was performed overnight at 4°C with gentle rocking in TBST buffer supplemented with 1% nonfat dry milk. On the next day, the membrane was washed three times for 10 min with 10 ml TBST buffer with gentle rocking and then incubated with a horse radish peroxidase coupled secondary IgG-specific antibody in TBST buffer supplemented with 1% nonfat dry milk for 1 h at RT with gentle rocking. After three additional washes for 10 min with 10 ml TBST buffer with gentle rocking, the membrane was developed using Immobilon Crescendo Western HRP Substrate (WBLUR0500; Millipore) and imaged on an Odyssey Fc imaging system (Model: 2800; LI-COR). After imaging, the membrane was stained with Methylene Blue to show the presence of DNA.

Genomic DNA was extracted using the QIAamp DSP DNA minikit (Qiagen, Hilden, Germany). A first batch of three isolates was processed as described in Rothen et al. (2017) (link). For a second batch of 15 isolates, paired-end libraries constructed by the Nextera XT DNA library prep kit (Illumina, San Diego, CA, United States) were sequenced on a MiSeq system (Illumina) using a 600-cycle MiSeq reagent kit v3 (Illumina). De novo assemblies were created using SeqMan NGen from the Lasergene genomics package version 12.1.0 (DNAStar, Madison, WI, United States) with standard settings. Comprehensive WGS information including accession numbers are provided in Supplementary Table S1.

To perform quantitative real-time PCR for CMV, DNA was extracted from whole blood using a QIAamp DSP DNA Mini Kit (Qiagen, Germany) and QIAcube (Qiagen, Germany) in accordance with the manufacturer’s instructions. Real-time PCR for CMV-DNA was performed using the LightCycler 480 (Roche Diagnostics, Germany) and Bio-Core CMV Quantification real-time PCR kit (Bio-Core, Korea). For the standardization of the results, the World Health Organization International Standard for human CMV for nucleic acid amplification techniques was used. The data were reported as copies/mL.

Genomic DNA was extracted from peripheral blood leukocytes using the QIAamp DSP DNA Mini Kit following the manufacturer's instructions (QIAGEN GmbH, Hilden, Germany). Two single nucleotide polymorphisms (rs429358 for codon 112 and rs7412 for codon 158) in the APOE gene were genotyped using LG AdvanSure^TM apoE Genotyping real-time PCR (LG Lifescience, Korea) on a SLAN real-time PCR Detection System (LG Lifescience, Korea) according to the manufacturer's instructions. Subjects with at least one APOE ε4 allele were identified as ε4 carriers. In addition, subjects with ε2/ε2, ε2/ε3, and ε3/ε3 alleles were identified as ε4 non-carriers.

Whole genome sequencing of all the study isolates was done using previously described methods (Pintara et al., 2018 ).
QIAamp DSP DNA mini kit (Qiagen, Germany) was used to extract DNA from the isolates using the QIAsymphony SP, following the Tissue HC 200 V DSP protocol. Quantitation of extracted DNA was done on a plate reader using the Quant-IT kit (Thermo Fisher Scientific, United States).
The Nextera XT DNA library preparation kit (Illumina, United States) was used for library preparation of the DNA samples. The DNA samples were then sequenced on the NextSeq500 (Illumina, United States) using the NextSeq 500 Mid Output V2 kit (Illumina, United States).
Trimmomatic v0.36 was used to trim sequence reads from the sequenced isolates and quality checked by FastQC v0.11.5 (Andrews, 2014 ; Bolger et al., 2014 (link)). Trimmed reads were then de novo assembled using the SPAdes assembler version 3.9.1 (Bankevich et al., 2012 (link)) and assemblies annotated using Prokka (Overbeek et al., 2014 (link)).