Pgem t easy vector systems kit

Viral cDNA sequencing of the Influenza A viruses was performed using freshly cultured isolates from the clinical specimens or nucleic acids from clinical samples. To analyze the phylogenetic relationships between the HA of the isolated pdm H1N1 and H3N2 serotypes, the HA and M genes were first examined via conventional RT-PCR using primers and a protocol as previously described [16 (link), 17 (link)]. Viral RNA was extracted from the freshly cultured isolates using an automated TANBead system (Taiwan Advanced Nanotech, Inc., Taiwan) or the QIAamp® Viral RNA Kit (Qiagen, Hilden, Germany). Further, the RT-PCR products were purified using a High Pure PCR Product Purification Kit (Roche Diagnostics, Mannheim, Germany) and cloned into the TA plasmid vector using a pGEM®-T Easy Vector Systems kit (Promega, Madison, WI, USA).
Plasmid DNA sequencing for the entire length of the cloned gene was performed using an ABI Prism 3730 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Two or three different plasmid DNAs of the same Influenza A virus isolate were sequenced to determine the accuracy of the gene sequencing. The gene sequences were then assembled using the Basic Local Alignment Search Tool from the National Center for Biotechnology Information [18 (link)].

Bisulfite treatment was performed with EZ RNA methylation Kit (Zymo Research) according to the manufacturer’s protocol. The target region of Sox9 was amplified by PCR using normal primers for untreated mRNAs and specific primers for bisulfite-treated mRNAs. To facilitate sequencing, the purified PCR products were ligated to the T vector using the pGEM-T easy vector systems kit (Promega). The ligation reaction was carefully transferred to the JM109 High Efficiency Competent Cells (Biomed), and cultured onto LB/ampicillin/IPTG/X-Gal plates. White colonies (at least 20 replicates for each sample) were selected for Sanger-based sequencing. The primers for the candidate fragment are shown in Supplementary Table 1.