Accuscript high fidelity 1st strand cdna kit

A QIAamp Viral RNA-mini kit (Qiagen) was used to extract RNA from liberated virions in the media of Vero E6 and Neuro-2a cells that had been inoculated with tick extract. Similarly, RNA was also extracted from Influenza B virus particles in the media of MDCK cells infected with that virus (non-template control), and from the spent media of non-inoculated cells of the same type grown in parallel under the same conditions (negative control). The RNA extracts were immediately stored at −80°C until cDNA could be generated using Accuscript High Fidelity 1^st strand cDNA kit (Agilent Technologies, Santa Clara, CA, USA) using both random 9-mers and TCRV-1 primer (see S2 Table for primer sequence).

Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen, Germany). The RNA concentration was determined using a spectrophotometer (MaestroNano, MaestroGen, Taiwan) and its integrity was assessed by 1.2% agarose-formaldehyde gel electrophoresis. First strand cDNAs were synthesized using Accuscript High Fidelity 1st strand cDNA kit (Agilent Technologies, United States). Real-time quantitative PCR was performed using QuantiSpeed SYBR Hi-Rox Kit (PhileKorea, South Korea) in a StepOnePlus Real-Time PCR System (Applied Biosystems, United States) as described previously (Lee and Kim, 2019 (link)). The primers used for qPCR are shown in Supplementary Table S1. The housekeeping gene 18S rRNA was used as an endogenous internal control to normalize gene expression. For northern blot analysis of ELIP3, the DNA probe was directly amplified and labeled with DIG-dUTP by PCR of the ELIP3 genes from cDNA using the DIG probe Synthesis Kit (Roche, Germany). The primers used for probe synthesis are shown in Supplementary Table S1. Northern blotting was performed as described previously (Han and Kim, 2013 (link)). The relative mRNA levels were quantified using Quantity One software (Bio-Rad, United States) and normalized by band intensities of rRNA.

Aliquots (300 μl) of 20 out of the 25 plasma samples for which virus was not isolated in cell cultures were treated with cyanse nuclease as mentioned above, and nucleic acids subsequently extracted using a QIAamp Viral RNA Mini Kit (Qiagen Inc.). Thereafter, Accuscript High Fidelity 1st strand cDNA kit (Agilent Technologies, Santa Clara, CA) primed with non-ribosomal hexamers (0.6 mM) [14 (link),26 (link)] in the presence of SUPERase-In RNase inhibitor (Ambion, Austin, TX) was used for cDNA synthesis [13 (link)]. PCR was thereafter performed with One Taq DNA polymerase (New England Biolabs) again primed with non-ribosomal hexamers. The process resulted in the production of PCR amplicons as evidenced on a 2% agarose gel stained with EtBr, and these were purified (Qiagen QIAquick PCR purification kit), then A-tailed with Taq DNA polymerase (New England Biolabs) as mentioned above. The A-tailed PCR amplicons were subsequently TA-cloned using a pCR2.1 vector with one shot TOP10 chemically competent E. coli cells, and the inserts (5 to 40 clones from each plasma sample, depending on how many TA clones were attained per plasma sample) were Sanger sequenced.