title: GSM4980217: miCLIP2 macrophages replicate 3; Mus musculus; OTHER
library_construction_protocol: RNA was extracted using Trizol (Thermo Fisher Scientific, 15596018) according to the manufacturer's instructions. Poly(A)+ RNA was extracted by incubating 100 µg total RNA with 200 µl Dynabeads solution (Dynabeads mRNA Direct Purification Kit, Thermo Fisher Scientific, 61012) and purified following manufacturer's protocols. 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.