GSM6806649: siWTAP.Input; Homo sapiens; OTHER
Total RNA was extracted form treated cells using RNAiso reagent (Takara, 9109) and then incubated with DNase I (Roche, 04716728001) to remove DNA contamination. Total RNA (200 μg) was randomly fragmented into ~200 nucleotides by RNA Fragmentation Buffer (100 mM Tris-HCl, 100 mM ZnCl2 in DEPC-treated water). Protein A magnetic beads (Thermo Fisher, 10002D) were pre-incubated with 5 μg of anti-m6A polyclonal antibody (Synaptic Systems, 202003), and then incubated with fragmented RNA in IP buffer (150 nM NaCl, 10mM Tris-HCl, 0.1% IGEPAL CA-630 in DEPC-treated water). The fragmented RNA binding with Protein A beads-antibody mixture was isolated using RNeasy Mini Kit (QIAGEN, 74106) according to the manufacturer's instruction. The library preparation for next-generation sequencing was constructed by TruSeq Stranded mRNA Sample Prep Kit (Illumina, USA) and quantified by BioAnalyzer High Sensitivity DNA chip.