FLAG-tagged hnRNPM iCLIP in AGS cells

We performed hnRNPM iCLIP to identify the potential targets. Overall design: Briefly, 5,000,000 AGS cells expressing FLAG-hnRNPM were ultraviolet crosslinked to a total of 0.4 J/cm2 in a UV cross-linker and harvested in ice-cold lysis buffer (10 mM HEPES (pH 7.4), 200 mM NaCl, 30 mM EDTA, 0.5% Triton-X 100, 1.5 mM DTT and 1× Protease-inhibitor cocktail (Roche, 04693116001)). Then, cells were sonicated for 5 min with a Sonics Vibra-Cell, and the cell supernatant was collected after centrifugation at 12,000 g for 15 min at 4 °C. The hnRNPM-binding complex was isolated by anti-FLAG (Sigma, F1804) coupled with Protein G Dynabeads (Life Technology, 10004D) and partially digested with 0.1U/µl RNase I (Thermo Scientific, EN0601) for 3 min at 37 °C. After washing once with lysis buffer, twice with high salt lysis buffer (10 mM HEPES (pH 7.4), 500 mM NaCl, 30 mM EDTA, 0.5% Triton-X 100, 1.5 mM DTT, 50 U/ml RNasin Inhibitor (Promega, N2111SV) and 1× Protease-inhibitor cocktail), RNAs were dephosphorylated at the 3' ends by T4 polynucleotide kinase (NEB, M0201L) for 20 min at 37 °C and then ligated at their 3' ends to a preadenylated DNA linker (5'-rApp-AGATCGGAAGAGCACACGTCTGAAC/AzideT/CCAGTCAC-3') with T4 RNA ligase I (NEB, M0204) for 60 min at 25?°C. The IR800CW dye conjugated to the NHS-Azide modification (/AzideT/) could be visualized using near infrared imager (Kaczynski et al., 2019). SDS-PAGE separation, visualization, protein-RNA complexes isolation, proteinase K treatment, and overnight RNA precipitation were performed as previously described (Kaczynski et al., 2019). For iCLIP cDNA library preparation, the isolated RNAs were reverse transcribed with barcoded primers. The cDNA library was purified by PAGE, circularized by CircLigase II (Lucigen, CL9021K), PCR-amplified by NEBNext Ultra II Q5 Master Mix (NEB, M0544S) for ~25 cycles and then subjected to high-throughput sequencing using an Illumina HiSeq2000 platform with a 50-nt run length.