GSM4980031: miCLIP C643 WT rep1, technical rep2; Homo sapiens; OTHER
C643 cells were grown on 15 cm dishes to approximately 90-95% confluence. Subsequently, the DMEM was discarded and the cells were washed twice with PBS. Total RNAs were extracted by using TRIzol reagent (Thermo Fisher Scientific), according to the manufacturer's instructions. Prior to isolation of polyadenylated RNAs (polyA(+) RNA), the total RNA samples were treated with DNase I (NEB), according to the manufacturer's protocol and subsequently cleaned up again by using TRIzol LS (Thermo Fisher Scientific). Approximately 5 µg of polyA(+) RNA was purified from 500 µg Total RNA by using the Dynabeads mRNA purification Kit (Thermo Fisher Scientific) for two rounds, according to the manufacturer's protocol. Briefly, 1 µg of polyA selected and fragmented RNA was incubated with an m6A antibody [Synaptic Systems, 202 003] and crosslinked twice at 150 mJ/cm² at 254 nm followed by immunoprecipitation with protein A Dynabeads. After stringent washing, the 3' end of the RNA was dephosphorylated using T4 PNK and subsequently pre-adenylated L3-App linker was ligated. The 5' end was radioactively labelled and the samples were purified by SDS PAGE and transferred to a nitrocellulose membrane. After exposure to a Fuji film, the protein-RNA complexes were isolated and elution of the RNA was accomplished by proteinase K treatment, phenol-chloroform extraction and ethanol precipitation. Reverse transcription was perform for cDNA synthesis using RT primers against the L3-App linker. The cDNA was purified using MyONE Silane bead clean up system and the 5' linker was ligated. A pre-amplification PCR with 6 cycles was performed and subsequently the samples were size-selected using ProNex beads. To prevent overamplification of the library, the PCR cycles were optimised to a minimum. For the preparative PCR, the minimum amount of PCR cycles was used followed by a second size selection step using ProNex beads. Libraries were profiled in a High Sensitivity D1000 ScreenTape on a Tape station 2200 (Agilent) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies). All samples in an experiment were pooled in equimolar ratio and sequenced on one NextSeq 500 High Output Flowcell, 75 cycle kit.