Splint‐ligation analysis

RNA was extracted with phenol:chloroform:isoamyl alcohol [25:24:1 (vol/vol)] followed by ethanol precipitation. To measure the total EBER1 RNA, 50 μg of total RNA was treated with alkaline phosphatase (calf intestine, CIAP) (Takara) for 1 h at 37°C according to the manufacturer’s recommendation, and then, 5 μg of CIAP‐treated RNA was phosphorylated by T4 polynucleotide kinase. The splint ligations were performed as previously described. For ligation, each reaction consisted of 100 fmol bridge oligonucleotide, 200 fmol FAM‐labeled ligation oligonucleotide, 5 μg untreated (for 5ʹ‐pRNA) or treated RNA (for total RNA), 1× T4 DNA ligase reaction buffer (Takara), and 10 units T4 DNA ligase (Takara). After the reaction mixture was denatured at 95°C for 1 min, cooled to 65°C for 5 min, and incubated at 37°C for 10 min, T4 DNA ligase was added to the reaction mixture and incubated at 30°C for 4 h. The reactions were terminated by heat inactivation at 75°C for 10 min and subsequently separated using denaturing 8 M urea 15% polyacrylamide gels. The images were obtained using a Bio‐Rad Molecular Imager^® Gel Doc XR System and analyzed using ImageJ software.