Dna rna shield solution

WNV-RNA positive plasma of the blood donor was provided by the Blood Service for Vienna, Lower Austria and Burgenland of the Austrian Red Cross. For PCR, pathogens in the sample were inactivated by immediately adding DNA/RNA Shield solution (Zymo Research, Irvine, USA) in the proportion 1:4 as described by [8 (link)], and stored at -20°C until further processing. The original plasma sample was independently investigated in the Czech laboratory for the presence of neutralizing antibodies against WNV strain Eg-101 by plaque-reduction neutralization test (PRNT) as described previously [9 (link)] and it was also used for virus isolation attempts (see below section “Virus isolation”).

We used ~10 whole adult DGRP379 and DGRP732 males and females. Flies were homogenized with an electric pestle in DNA/RNA Shield solution (Zymo Research). Homogenized tissue was digested with Proteinase K and RNA was purified with the Zymo Quick-RNA Plus Kit (Zymo Research). Ribosomal RNAs were removed using siTools rRNA depletion Kit (Galen Laboratory Supplies) and MyOne Streptavidin C1 Dynabeads (ThermoFisher) (#65001). Ribosomal RNA-depleted RNA was purified using the RNA Clean and Concentrator-5 kit (Zymo Research). Illumina libraries were generated using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB).
RNA sequencing reads were first aligned to the FlyBase r6.27 rRNA sequences using HISAT2 [77 (link)]. Non-ribosomal sequences were subsequently aligned to FlyBase r6.27 transcript sequences using htseq-ct [78 (link)]. Counts were filtered to include only expressed transcripts using DESeq2 [79 (link)] (rowSums(DESeqDataSetFromHTSeqCount) > = 1), which were subsequently normalized using rlog transformation (blind = TRUE) in DESeq2 [79 (link)].

Swab sampling of both cats, Cat 1 and Cat 2, and their environment was carried out on 16 and 19 November 2020. Cotton swabs with plastic shafts (Lidl, Weinfelden, Switzerland and Heinz Herenz, Hamburg, Germany) that were separately sealed in reclosable plastic bags (Minigrip^® Red line, Alpharetta, GA, USA) were used for sampling. Strict caution was taken to prevent contamination of the swab sampling area during collection.
From each cat, oropharyngeal, nasal, and rectal swabs were collected. Additionally, swabs from the fur and the surface of the cats preferred sleeping spot were taken to look for environmental and surface contamination. After sampling, the swab tip was stored in a previously labeled 1.5 mL screw cap tube (Sarstedt AG and Co. KG, Nümbrecht, Germany) prefilled with 300 µL of DNA/RNA shield solution (Zymo Research Europe GmbH, Freiburg, Germany), which also ensures nucleic acid stability during sample storage/transport at ambient temperatures and inactivates nucleases and infectious agents. The overlaying part of the shaft was cut with clean scissors and the tube was closed. The specimens were shipped to the Clinical Laboratory at ambient temperature, stored at 4 °C, and further analyzed within 48 h.

Microbial DNA enrichment and host DNA depletion was performed on pooled oesophageal brushings using the MolYsis Basic5 kit (Molzym, Bremen, Germany) according to the manufacturer’s instructions, with the resulting cell pellet stored at −20 °C until DNA extraction. Saliva samples were always collected prior to the collection of oesophageal brushings and stored at 4 °C in 1 : 1 DNA/RNA shield Solution (Zymo Research) for 24–48 h before DNA extraction. DNA was extracted from saliva and oesophageal brushings using the QIAmp DNA Mini Kit according to manufacturer’s instruction (Qiagen, Hilden, Germany), with DNA fractions eluted in 50 µl of dH₂O and stored at −20 °C. All oesophageal brush samples were processed within 1–3 h of collection.
DNA quantification was performed using a Qubit 3.0 fluorometer (Invitrogen, CA) and double-stranded DNA (dsDNA) HS assay kit. Pooled Illumina sequencing libraries were constructed according to methods previously described by Ravi and colleagues [21 (link)]. Paired-end metagenomic sequencing was performed on the Illumina Novaseq 6000 platform yielding 2×250 bp paired-end sequencing reads.

Stool samples were collected at home, stored under refrigeration, and transported within 24–48 h to our laboratory by the participants prior to visits at baseline, week 4, and week 12. Assembled stool preparation kits including gloves, sterile cotton swab sticks, and a stool container with DNA/RNA shield solution were provided to participants (Zymo Research). DNA isolation and quantification, and 16 rRNA gene sequencing were performed at Laragen Inc.

Blood samples were obtained from donors attending the blood bank at the Instituto Nacional de Enfermedades Respiratorias “Ismael Cosio Villegas”. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using a density gradient as the standard Lymphoprep. In PBMCs, a positive selection for CD14+ cells was performed using magnetic microbeads coated with an anti-CD14 monoclonal antibody (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity of the CD14+ fraction was analyzed by flow cytometry using anti-human CD14, CD2, and CD19 monoclonal antibodies from BioLegend. The efficiency of this process was regularly >90%. To promote the differentiation from monocytes to macrophages (MDMs), 2 × 10⁶ CD14+ cells were cultured in Costar 6-well plates in RPMI-1640 culture medium (GIBCO, Grand Island, NY, USA), supplemented with 2 mM L-glutamine (GIBCO, Grand Island, NY, USA), 100μg/mL streptomycin, 100 IU/mL penicillin, and 10% fetal bovine serum (FBS, GIBCO, Grand Island, NY, USA) for 7 days at 37 °C, 5% CO₂. Then, MDMs were lysed with 200 µL of lysis buffer (Pierce RIPA buffer; Thermo Scientific, Waltham, MA, USA) to evaluate proteins or suspended in DNA/RNA Shield solution (Zymo Research, Irvine, CA, USA) and stored at −70 °C until use for RNA extraction. More details of the antibodies used in this study are shown in Table A1 (Appendix A).

All N95 respirators were carefully bagged upon collection and transferred to the lab for testing. The mask outer layer was removed with the aid of sterile scissors, placed in a sterile petri dish and saturated with 2 ml of DNA/RNA shield solution (Zymo Research, Germany) (16 (link)). After 5 min, the mask outer layer was transferred to a 15 ml falcon using sterile forceps. The tube was centrifuged for 1 min at 3,300 g, the supernatant was recovered and stored at 4°C until DNA extraction.