Sequencing of RNA fragments immunoprecipitated by anti-Flag in ALKBH5 overexpression NOMO1 cells

To identify the direct mRNA targets of ALKBH5, we conducted RIP-seq with total RNA samples from ALKBH5 overexpression NOMO1 cells AML cells (herein is NOMO1) were transduced with lentivirus expressing ALKBH5 CDS with 3xFlag, then GFP positive cells were sorted out after puromycin selection. ALKBH5 overexpression cells were UV crosslinked then harvested for lysis. anti-Flag antibody (Sigma-Aldrich) was used to immunoprecipitate ALKBH5 protein binding RNAs. Immunoprecipitated samples were subjected to Proteinase K digestion (ThermoFisher). Total RNA was extracted from both input and immunoprecipitated RNA using TRIzol followed by Direct-zol RNA Miniprep (Zymo). RNA was then fragmented using the Bioruptor Pico sonication device. Libraries for high-throughput sequencing were constructed using the TruSeq Stranded mRNA Sample Prep Kit (Illumina), and were quantified by BioAnalyzer High Sensitivity DNA chip. RIP-seq libraries were sequenced on the Illumina HiSeq 4000 sequencing system.