GSM7917341: YTHDF2 iCLIP2, HEK293T, STM2457, 12h, rep4; Homo sapiens; RIP-Seq
In vivo iCLIP2 for YTHDF2 was performed as described before (Buchbender, A. et al 2020). Briefly, HEK293T cells treated with DMSO or STM2457 for 12 h were irradiated with 150 mJ/cm2 in a UV-C crosslinker (CL‑1000 Ultraviolet Crosslinker, UVP) at 254 nm. Crosslinked cells were lysed and RNA was digested to create 50–200 nt long fragments. YTHDF2 protein immunoprecipitation was performed with an YTHDF2-specific antibody (AVIVA SYSTEMS BIOLOGY, ARP67917_P050) conjugated to protein A beads. Protein-RNA complexes were visualized using 5' end radioactive labelling of the precipitated RNA, followed by SDS-PAGE and transfer to a nitrocellulose membrane. Protein-RNA complexes were recovered from the membrane and treated by proteinase K to get protein-free RNA. cDNA was generated by reverse transcription, with termination occurring at the crosslinked site, thereby generating truncated cDNAs. MyONE beads were used to clean cDNA twice and the library was subsequently subjected to PCR amplification and ProNex size selection for amplification and purification, respectively. iCLIP libraries were sequenced on an Illumina NextSeq 500 sequencing device with 77 nt single-end reads