Klenow fragment polymerase

RNA was extracted from the isolated virus using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). Reverse transcription and polymerase chain reaction (PCR) were performed using the SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and a random primer containing a 20-base arbitrary sequence at the 5′ end followed by a randomized octamer (8N) at the 3′ end. A single round of priming and extension was performed using the Klenow fragment polymerase (New England Biolabs, Ipswich, UK). PCR amplification with a primer consisting of only the 20-base arbitrary sequence of the random primer was performed with 20 cycles of 94 °C for 15 s, 60 °C for 30 s and 68 °C for 1 min and a final extension at 68 °C for 7 min in an automated thermal cycler (Applied Biosystems, Carlsbad, CA, USA). Standard precautions were taken to avoid PCR contamination, and no amplified PCR product was observed in the negative control. The PCR product was purified using the MinElute PCR Purification Kit (Qiagen) following the manufacturer's protocol. The purified DNA was eluted in 15 μL of EB buffer and used as the template for library construction.^{16 (link)}

The QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) was used to extract viral RNA from the culture supernatants of infected Vero cells. Reverse transcription and PCR were performed using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) using primer contained a 20-base arbitrary sequence at the 5′ end followed by a randomized octamer (8 N) at the 3′ end as described previously [16 (link)]. A single round priming and extension was performed using Klenow fragment polymerase (New England Biolabs, Ipswich, MA, USA). PCR amplification were performed in an automated thermal cycler (Applied Biosystems, Foster City, CA, USA) with primer consisting of only the 20-based arbitrary sequence of the random primer with 20 cycles of 94 °C for 15 s, 60 °C for 30 s and 68 °C for 1 min and a final extension of 68 °C for 7 min. Standard precautions were taken to avoid PCR contamination and no amplified PCR product was observed in negative control. The PCR product was purified following the manufacturer’s protocol using the MinElute PCR Purification Kit (Qiagen, Germany) with slight modification. The purified DNA was eluted in 15 μL of EB buffer and used as the template for library construction.

A brain sample was fragmented and passed through an insulin needle. The homogenate was clarified by centrifugation and filtered with a 0.45 µm syringe filter. The filtrate was digested with Turbo DNase (Ambion) and RNase I (Invitrogen) to remove non-protected human nucleic acids. Remaining nucleic acids were extracted using the QIAamp viral RNA minikit (Qiagen). Purified RNA was reverse-transcribed with Superscript III reverse transcriptase (Invitrogen) and second-strand DNA synthesis was performed with Klenow fragment polymerase (New England Biolabs) using barcoded primers consisting of a 20-nucleotide–specific sequence upstream of a random nonamer, as previously described [18 (link)]. The resulting DNA products were PCR amplified, and libraries were constructed with the Nextera XT DNA library preparation kit (Illumina) and sequenced on a NextSeq Illumina platform (2 × 150 bp run).