| Summary: | The expression frequency of the three orthologous gene was analyzed based on the read number obtained by sequence analysis of mRNA using MiSeq. Total RNA was isolated from cells using ISOGEN (Wako) according to the manufacturer’s instructions as follows. Cells were ground in liquid nitrogen to a fine powder, and approximately 3 g was mixed with 15 ml ISOGEN solution, followed by the addition of 3 ml chloroform. The mixture was left at room temperature for 3 min and centrifuged at 2,300 g for 20 min. The supernatant was transferred to a fresh tube and mixed with 7.5 ml isopropanol. The mixture was left at room temperature for 10 min and centrifuged at 2,300 g for 20 min. The pellet was dried and dissolved in 300 μl of RNase-free water. mRNA was purified from approximately 200 μg total RNA using an OligotexTM-dT30 mRNA Purification kit (Takara, Shiga, Japan) according to the manufacturer’s instructions. The library was prepared with TruSeq Stranded mRNA Library Prep (Illumina Inc., San Diego, CA, USA), and obtained the sequences with MiSeq (Illumina) according to the manufacturer’s instructions. Read number of each nucleotid was viewed with IGV [ 30 ]. (TIF)
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