GSM7847042: HUDEP2_RIP_WT_input; Homo sapiens; RIP-Seq
The lentiviral vector TRE-FAM46C-strep was introduced into E6E7 cells through transfection, followed by selection with puromycin for a duration of 3 days. After a 24-hour induction with Doxycycline (Dox), a total of 2 million cells were collected for RNA immunoprecipitation (RIP) analysis. For the purpose of formaldehyde crosslinking, the cells were first centrifuged and then resuspended in 500 μl of phosphate-buffered saline (PBS) containing 1.5 μl of 37% formaldehyde, resulting in a final concentration of 0.1%. The cells were then incubated at room temperature for 10 minutes. Subsequently, the crosslinking reaction was quenched by the addition of 37.5 μl of 2M glycine, and the cells were incubated for an additional 10 minutes at room temperature. Following this, the cells were centrifuged at 500g for 5 minutes, and the resulting cell pellet was resuspended in 500 μl of buffer A (10mM HEPES, pH 7.9, 10mM KCl, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, 1x proteinase inhibitor, RNase inhibitor) on ice for a duration of 15 minutes. To this suspension, 34.4 μl of 10% NP40 was added, and the mixture was vortexed for 1 minute. The samples were then subjected to centrifugation at 6000g, 4℃ for 5 minutes. Next, Strep beads (IBA, 2-4090-002) were washed three times with wash buffer (10 mM Tris/HCl pH 8.0, 150 mM NaCl, 0.5 mM EDTA) and then resuspended in the supernatant of the samples. Additionally, 15 μl of 5M NaCl was added to the suspension, which was subsequently incubated at 4℃ for 2 hours on a roller. After the incubation period, the supernatant was removed from the samples, which were then subjected to four washes in wash buffer (300 mM NaCl) using a magnetic rack. Subsequently, 50 μl of biotin elution buffer (100 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 50 mM biotin) was added to the samples and incubated for 10 minutes at room temperature. The elution was mixed with 250 μl of proteinase K buffer and 4 μl of 20 mg/ml proteinase K, and incubated overnight at 65℃. RNA extraction was then carried out using TRIZOL. For UV-RIP analysis, cells were subjected to UV crosslinking at 254nm, 4000 (400mJ/cm2) in cold PBS, with the remaining steps being similar to the aforementioned protocol. RNA was used to construct an RNA Next-Generation Sequencing (NGS) library using the TruePrep RNA Library Prep Kit for Illumina (vazyme, TR503-01) before sequencing.