Bio 16 utp

The MEGAscript™ T7 High Yield Transcription kit (Invitrogen, USA) was used to transcribe biotin-labelled RNAs in vitro. Bio-16-UTP (10 mM, Ambion) was used for transcription. After adding 2 µL of Dnase I, the Eppendorf tube was incubated at 37 °C for 15 min to remove the DNA, then 2 µL of 0.2 M EDTA (pH 8.0) was added. In order to allow the RNA to form secondary structure, 1 µg of biotinylated RNA in RNA structure buffer was heated at 95 °C for 2 min, put on ice for 3 min, then left at room temperature for 30 min. Magnetic beads (Invitrogen, USA) were used to bind and enrich the RNAs. Folded RNA was then mixed with cytoplasmic extract from liver cancer cells in 500 µL RIP wash buffer. The Magnetic beads were re-suspended in 50 µL RIP wash buffer, then the suspension was added to Dynabeads M-280 Streptavidin (60210, Invitrogen) and incubated at 4 °C. The suspension was centrifuged for 1 min, and the supernatant was discarded. Magnetic beads were washed briefly with RIP wash buffer for six times and boiled in SDS buffer. The retrieved proteins were detected by western blot and mass spectrometry. RNA probes were as follows: lnc-Ma301 sense: taatacgactcactatagggGGAGAGTTTGGGTCACAGGAGC, lnc-Ma301 antisense: CCTACTTGTTTTTTTTATTTTGG.

The half-life of IVS RNA in E. coli was determined using a RPA. The IVS element and ~400 bp of flanking sequences were PCR amplified by standard protocol using IVSflank_F and _R primers (Table S1) and cloned into pCR2.1-TOPO per manufacturer's instructions (Invitrogen) to generate pIVS1. E. coli TOP10F' was transformed with pIVS1 to generate strain IRW205, which was cultured in LBamp for 16 h at 37°C and then used to inoculate LBamp at a 1:20 dilution. Following growth for 2 h at 37°C, IPTG was added to 1 mM and cultures were allowed to grow an additional 2 h. Rifampin was added to a final concentration of 160 μg/ml and 1 ml samples were taken at 5 min intervals for 35 min. RNA was purified from samples using TRI Reagent (Ambion) and processed using a RPA III kit (Ambion) [3 μg total RNA; 800 pg IVS probe (Table S3)], as previously described (Hicks et al., 2011 (link)). Biotinylated IVS RPA probes were prepared using a MEGAScript kit as instructed (Thermo Fisher), biotin-labeled UTP (Bio 16-UTP; Ambion), and corresponding IVS primers (Table S1; IVSprobe_F and IVSprobe_R+T7).