GSM3073821: iCLIP-seq of Flag-GFP in N2A cells, replicate 2; Mus musculus; RIP-Seq
Cells were crosslinked (0.15 J/cm2) at 254 nm with a Stratalinker 1800. Three independent replicates were used for generating iCLIP samples. Lysates generated from the crosslinked cells were treated with Turbo DNAse (Ambion) and diluted RNAse I (Ambion) for 5 minutes at 37°C to digest the genomic DNA and trim the RNA to short fragments of an optimal size range. RNA-protein complexes were immunoprecipitated using 100 μL of protein G Dynabeads (Life Technologies) and 10 μg of anti-Flag (Sigma) antibody. Following stringent high salt washes, the immunoprecipitated RNA was 3´ end dephosphorylated and a pre-adenylated adaptor was ligated at the 3´ end as described for a previously described enhanced CLIP ligation method (Van Nostrand et al., Nat. Methods 2016) for adaptor ligation. After additional washes, the immunoprecipitated RNA was 5´ end-labeled using radioactive 32P isotopes. The immunoprecipitated complexes were separated with SDS-PAGE and transferred to a nitrocellulose membrane (Protran). RNA was recovered by digesting proteins using proteinase K. RNA was reverse transcribed into cDNA using reverse transcription primers that include barcode sequences to enable multiplexing,  a unique molecular identifier, and a BamHI restriction site. The cDNA was size selected (low: 70 to 85 nt, middle: 85 to 110 nt, and high: 110 to 180 nt), circularized to add the adaptor to the 5′ end, digested at the internal BamHI site, and then PCR amplified using AccuPrime SuperMix I (Life Technologies). The final PCR libraries were purified by agarose gel electrophoresis using gel extraction columns (QIAGEN), eluted DNA was mixed at a ratio of 1:5:5 from the low, middle, and high fractions and submitted for sequencing.
GEO Accession
GSM3073821