GSM7232728: PC3 cells, expressing HA-METTL1; Homo sapiens; RIP-Seq
Cells were cross-linked at 365 nm (150 mJ/cm2) inStratalinker 2400and then lysed with CLIP Lysis buffer (50 mM Tris-HCl pH7.4, 100 mM NaCl, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate in the presence of protease inhibitors) and passed 7-8 times through a 27G needle. For RNA digestion, two dilutions of RNase I (ThermoFisher Scientific, EN0602) were prepared in PBS (high: 1/50, and low: 1/250). 10mL of each dilution and 2mL of TurboDNase (ThermoFisher Scientific, AM2238) were added to 1 ml of each cell lysate (corresponding to ~1mg of protein) and incubated for 3 min at 37 ºC, and then placed on ice for > 3 min. For HA-METLL1 immunoprecipitation 30 µl of Pierce anti-HA magnetic beads (ThermoFisher Scientific, #88836) were used. RNA 3' ends were dephosphorylated usingPNK (NEB, M0201L) and FastAP Alkaline phosphatase (ThermoFisher Scientific, EF0654). Next, the 3' pre-adenylated adapters L3-IR-App were ligated withT4 RNA Ligase (NEB, M0437M). Adaptors were removed and RNAs were treated withRecJfendonuclease (NEB, M0264S). RNA-protein complexes were run on a NuPAGE4-12% Bis-Tris gel(ThermoFisher Scientific, #NP0322BOX)and transferred onto a nitrocellulose membrane. RNA-bound complexes between 75 and 130 KDa were cut out from the membrane, andtreated with proteinase K. After phenol extraction,RNAs were reverse-transcribed withSuperscript IV (ThermoFisher Scientific, #18091050), and cDNA was circularized withCircLigase II (Biosearch Technologies, #CL9021K).PCR was performed with P3/P5 Solexa primers for 22 cycles, and the appropriate size of products were confirmed by gel electrophoresis.   Sequencing of the libraries was performed on a HiSeq X instrument following standard manufacturer's procedures, with single-read, 151-bases read profile. PAR-CLIP