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RNA immunoprecipitation (RIP)

XJ Xi Jin
LG Li-Ping Ge
DL Da-Qiang Li
ZS Zhi-Ming Shao
GD Gen-Hong Di
XX Xiao-En Xu
YJ Yi-Zhou Jiang
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RIP was performed using a Magna RIP Kit according to the manufacturer’s instructions (Millipore). Briefly, 2 × 107 cells were trypsinized and rinsed twice with ice-cold PBS, resuspended in an equal pellet volume of RIP lysis buffer supplemented with a protease inhibitor cocktail and an RNase inhibitor and subjected to a single freeze-thaw cycle to gently lyse the cells. A total of 5 μg of antibody was added to the magnetic beads, and the mixture was incubated for 30 min at room temperature in RIP wash buffer with agitation. The beads were washed three times in RIP wash buffer, and the cell lysates were added and incubated at 4 °C overnight. Each immunoprecipitant was resuspended in proteinase K buffer (1.2 μg/μL proteinase K and 1% SDS) and was incubated at 55 °C for 30 min. RNA was isolated by phenol, chloroform and isoamyl alcohol according to the manufacturer’s instructions and was detected by qRT-PCR.

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