miCLIP-sequencing

Nuclear RNA from PANC-1 cells was isolated and fragmented. RNA precipitated by ethanol was diluted in 450 μl of RIP buffer and incubated with 15 μg of anti-m⁶A antibody at 4 °C for 1 h by rotating head over tail. The solution was then transferred into a 12-well cell culture plate and cross-linked twice with 150 mJ/cm² of UV light (254 nm). After cross-linking, the solution was transferred into Eppendorf tubes and incubated with 100 μl of Protein A/G beads (Millipore) overnight at 4 °C with rotating. Bead-bound antibody-RNA complexes were then recovered on a magnetic stand and washed with high-salt buffer (50 mM Tris, pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, and 0.1% SDS), immunoprecipitation buffer and polynucleotide kinase (PNK) wash buffer (20 mM Tris, 10 mM MgCl₂, and 0.2% Tween 20), respectively. RNA 3′ ends were dephosphorylated on beads with PNK (New England BioLabs) for 30 min in dephosphorylation buffer (70 mM Tris, pH 6.5, 10 mM MgCl₂, and 1 mM DTT). After another round of extensive washing (twice with PNK wash buffer, once with immunoprecipitation buffer, once with high-salt buffer, and twice with PNK wash buffer), the 3′ adaptor was ligated with T4 RNA ligase (New England BioLabs) overnight. The antibody-bound RNA was recovered by treatment with proteinase K, acidic phenol/chloroform extraction, and ethanol precipitation. Purified RNA was reverse transcribed with Superscript III reverse transcriptase (Life Technologies). First-strand cDNA was size-selected on an 8% TBE-Urea gel (Life Technologies), and the regions corresponding to 100–180 nucleotides were used for further analysis. After circularization and re-linearization of cDNA, libraries were PCR amplified for 18–21 cycles and sequenced on an Illumina HiSeq X Ten.