Kingfisher magnetic particle processor

For routine analysis, nasopharyngeal specimens in universal transport medium (UTM) were opened under biosafety hood, and 200 μL were used for RNA extraction using MagMAX Viral/Pathogen II (MVP II) Nucleic Acid Isolation Kit and the automated KingFisher magnetic particle processor (Thermo Fisher Scientific, Waltham, MA, USA), as indicated in the manufacturer’s instructions. Then, 10 μL of the extracted RNA underwent real-time reverse transcription polymerase chain reaction (qRT-PCR) using the TaqPath™ COVID-19 CE-IVD RT-PCR Kit assay (Thermo Fisher Scientific, Waltham, MA, USA) following the kit’s instruction. The QuantStudio 5 Real-Time PCR System assay (DX) was used for qRT-PCR analysis (Thermo Fisher Scientific, Waltham, MA, USA). The TaqPath™ assay targets three different viral genomic regions: ORF1ab, N and S genes. A valid negative result for SARS-CoV-2 was determined by amplification of the only MS2 internal control. A specimen was considered positive in the presence of amplification of at least two of the three target genes. Particularly, positive samples were classified as “S positive” when all the three targets were detected or when only two targets were detected, including S. On the other hand, samples were defined as “S negative” when the S target was not amplified.

For tissues, samples were homogenized with lysis buffer (RLT [Qiagen] + 1 %-β mercaptoethanol) using a rotor-stator homogenizer. RNA was extraced from tissue homogenate with KingFisher™ Pure RNA Tissue Kit (Thermo Scientific). Briefly, tissue homogenates were combined with magnetic beads and ethanol, and loaded onto a KingFisher Magnetic Particle Processor (Thermo Scientific). Samples were DNase-treated, washed, and eluted in RNase-free water. Due to the low level of total RNA isolated from dog sciatic nerve, the five individual samples were pooled and split into three sample replicates for analysis. RNA isolation of the bone marrow was performed with 3-fold volume of TriReagent LS (Life Technologies). Additional details are in the Additional file 1.

In the segregating F₂ population, DNA was isolated from 8mm young leaf discs using a mag Plant DNA Isolation Kit (AGOWA) in conjunction with a KingFisher magnetic particle processor (Thermo Scientific) according to manufacturer’s specifications. For all other experiments, DNA was extracted using a modified CTAB protocol. CAPS and size markers were developed to genotype BoFLC2 and BoFLC4-1. Details of these markers are shown in Supplementary Table S2.

Immunoprecipitation (IP) of conditioned cell media from transiently transfected HEK293T cells was performed using the method described previously with minor modifications [41 (link)]. In brief, 4 µg of antibodies 6E10 and 4G8 (BioLegend) were conjugated separately with Dynabeads M-280 Sheep Anti-Mouse IgG (Invitrogen) through an incubation period of one hour at room temperature. Phosphate buffered saline (PBS) was used as negative control and pooled human CSF was used as positive control. Cell media samples and the control samples were incubated overnight at 4 °C with 50 µl of the antibody-conjugated beads in the presence of 0.2% (w/v) Triton X-100 (Sigma Aldrich). King Fisher magnetic particle processor (Thermo Scientific) was used for automated elution of Aβ peptide consisting of sequential washes in 0.2% Triton X-100, PBS and 50 mM ammonium bicarbonate. The final eluates were collected in 100 µl 0.5% (v/v) formic acid (Fluka), dried in a vacuum centrifuge and kept at − 80 °C.

RNA was extracted using a MagMAX-96 AI/ND Viral RNA Isolation Kit (Applied Biosystems, Foster City, CA) using a KingFisher Magnetic Particle Processor (Thermo Scientific, Waltham, MA). RNA extracts were screened for IAV using the AgPath-IDTM One Step RT-PCR mix (Applied Biosystems, Foster City, CA) and an ABI 7500 real-time PCR System (Applied Biosystems, Foster City, CA). The real-time reverse transcription polymerase chain reaction (RRT-PCR) targets a conserved region of the IAV matrix gene [11 (link)]. Each RNA extract was tested in one replicate. Each RRT-PCR plate included an IAV isolate from cell culture as a positive control and VTM diluent as a negative control. Samples with a cycle threshold (Ct) value <45 were considered positive.

Fecal swabs collected with Synthetic-Tipped Applicators (Thermo Fisher Scientific, Waltham, MA) were re-suspended by being swirled in 1 ml PBS (Thermo Fisher Scientific, Waltham, MA), followed by total genetic materials extraction using MagMAX™ viral RNA isolation kit (Life Technologies, Carlsbad, CA) on a Kingfisher magnetic particle processor (Thermo Fisher Scientific, Waltham, MA) following the manufacturer’s instructions with minor modification as described previously [2 (link)]. Specifically, extracted DNA/RNA was eluted into 50 μl elution buffer. Extracted nucleic acids were then treated by RNase-free DNase I (Qiagen, Hilden, Germany) for 15 min at ambient temperature to remove DNA, and was then purified by Agencourt® RNAclean® XP Beads (Beckman Coulter Life Sciences, Indianapolis, IN) following the manufacturer’s instruction.