Quantifluor one dsdna system kit

RNA from the samples that tested positive and showed a Ct value < 31 on real-time RT–PCR was analyzed by WGS and phylogenetic analysis. Total RNA was treated with TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 20 min and then purified by RNA Clean and Concentrator-5 Kit (Zymo Research, Irvine, CA, USA). RNA was used for the assessment of sequencing independent single primer amplification protocol (SISPA) using SuperScript^® IV Reverse Transcriptase (Thermo Fisher Scientific, Waltham, MA) and a combination of two primers including the random-tagged primer FR26RV-N 5′-GCCGGAGCTCTGCAGATATCNNNNNN-3′ and the poly-A tagged primer FR40RV-T 5′-GCCGGAGCTCTGCAGATATCTTTTTTTTTTTTTTTTTTTT-3′ [26 (link)]. The PCR product was purified by ExpinTM PCR SV (GeneAll Biotechnology CO., Seoul, Korea) and then quantified using the QuantiFluor One ds DNA System kit (Promega, Madison, WT, USA). Libraries were prepared using Nextera DNA Flex Library Prep (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s protocol. Deep sequencing was performed on the MiniSeq (Illumina Inc., San Diego, CA, USA) by the MiniSeq Mid Output Kit (300-cycles) and standard 150 bp paired-end reads.

Total DNA was isolated from FFPE samples using the Maxwell^® RSC DNA Formalin-fixed paraffin embedded Kit (Promega, Madison, WI, USA) and a Maxwell^® RSC Instrument (Promega). DNA from frozen tissue was extracted with DNeasy^® Blood and Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocols. In FFPE, at least 2 cores were obtained from selected tumor areas. DNA was quantified using QuantiFluor^® ONE dsDNA System kit (Promega) or Quant-iT^TM PicoGreen^TM dsDNA protocol (Invitrogen, Carlsbad, CA, USA).

Total genomic DNA was isolated using the DNA-sorb-V Kit (Central Research Institute for Epidemiology, Russia) in accordance with the manufacturer’s instructions. DNA concentration was measured on a Quantus fluorimeter (Promega, United States) using the QuantiFluor^®ONE dsDNA System Kit (Promega, United States).