GSM7118169: Hep3B KD, RIP-seq, input; Homo sapiens; RIP-Seq
Cells were washed twice with ice cold PBS, cells were homogenized in adequate volumes of polysome lysis buffer (10 mM HEPES-KOH [pH 7.0], 100 mM KCl, 5 mM MgCl2, 25 mM EDTA, 0.5% IGEPAL, 2 mM  dithiothreitol [DTT], 50 U/ml RNase OUT [Life Technologies, NY, USA], 1× complete protease inhibitor tablet [Roche]). The suspension was centrifuged at 14,000 g at 4°C for 10 min to remove debris. and then immunoprecipitated with Protein A+G Magnetic beads coupled with YTHDF1 antibody, then washed five times with NT2 buffer (50mM Tris-HCl pH 7.4，150 mM NaCl，1mM MgCl2，0.5% IGEPAL, 2 mM dithiothreitol [DTT], 50 U/ml RNase OUT [Life Technologies, NY, USA], 1× complete protease inhibitor tablet [Roche]), and treated with proteinase K. RNA was extracted using Trizol (thermo). RNA immunoprecipitated RNA and input RNA use KAPA RNA HyperPrep Kit with RiboErase (HMR)