| attributes |
| sample_name |
DRS001612 |
| sample comment |
This sample is total RNA that was not treated BRIC in the UPF1-depleted HeLa cells.It was harvested in 0h time point.Sequencing analysis was carried out without polyA selection.The siRNA duplexes were used at a final concentration of 10 nM and were transfected into cells using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s instructions. BRIC was performed basically as described. In brief, cells were incubated at 37°C in the presence of 150 μM 5'-bromo-uridine (BrU)for 24 h in a humidified incubator with 5% CO2. At indicated time points after the replacing of BrU-containing medium with BrU-free medium, cells were harvested for RNA preparation using RNAiso Plus, and total RNA was isolated. Twelve micrograms of BrU-labeled total RNA was denatured by heating at 80°C for 1 min and then added to the anti-BrdU mAb-conjugated beads, which contain 2 μg of the anti-BrdU mAb (clone 2B1, MBL). The mixture was incubated at room temperature for 1 h with rotation. Beads were washed 3 times with 0.1% BSA in PBS, and ISOGEN LS was added, followed by RNA isolation in accordance with the manufacturer’s instructions. The isolated RNA was used for RT-qPCR or deep sequencing. |
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