Illumina HiSeq 4000 sequencing; iCLIP of Dazl in mouse embryonic stem cells
iCLIP of Dazl in mouse embryonic stem cells
Mouse embryonic stem cells were UV cross linked and lysed in 50 mM TrisвЂ“HCl, pH 7.4, 100 mM NaCl, 1% Igepal CA-630, 0.1% SDS, 0.5% sodium deoxycholate and ROCHE c0mplete protease inhibitor cocktail (1x) RNA cross-linked to DAZL was immunoprecipitated and PAGE was performed on protein-RNA complexes. After transfer of the protein-RNA complexes to nitrocellulose (wet blotting) the complexes were digested with Proteinase K and the liberated RNA-peptide complexes purified using PCA.  Library construction was carried out according to the iCLIP protocol described elsewhere [1]. RNA was ligated with a IRL3 3'adapter, circularised and reverse transcribed using barcoded RT primers.  [1] Blazquez L et al. Mol Cell 2018. doi: 10.1016/j.molcel.2018.09.033