We crosslinked 6 hour doxycycline-induced pSM33 mouse male ES cells with 0.4 J/cm2 of UV254. Cells were lysed and RNA was digested with RNase I to achieve a size range of 100-500 nucleotides in length. For each CLIP sample, target proteins were immunoprecipitated from 20 million cells with antibody pre-coupled with Protein G beads. Samples were then washed and RNAs were eluted with NLS elution buffer with 100 mM DTT. Samples were then run through a standard SDS-PAGE gel and transferred to a nitrocellulose membrane, and a region 75 kDa above the molecular size of the protein of interest was isolated and treated with protease followed by phenol/chloroform/isoamyl alcohol extraction to isolate the RNAs. The purified RNA in each sample was dephosphorylated and ligated to a mixture of barcoded adapters in which each adapter had a unique barcode identifier and Illumina sequencing libraries were generated according to our Massively Multiplexed RNA Sequencing method.
GEO Accession
GSM2299085