GSM1204907: E14.HA-EZH2.HA.b1; Mus musculus; RNA-Seq
Whole cell extracts were obtained by incubating the cells for 10 min at 37C in an appropriate volume of CLIP buffer (20 mM HEPES pH 7.4, 5 mM EDTA, 150 mM NaCl, 2% lauryldimethylbetaine) supplemented with protease inhibitors, 20 U/ml Turbo DNase (Life technologies), and 200 U/ml murine RNase inhibitor (New England Biolabs). [Immunoprecipitation and size selection protocol] After clearing the lysate by centrifugation, tandem affinity purification was performed, first with streptacting macropreps (3h, 4C), then elution with 2 mM biotin and finally an overnight IP with HA antibodies. The extracts were treated with various concentration of RNAse A + T1 cocktail (Ambion) for 5’ at 37C. Immunocomplexes were recovered by adding protein G-coupled dynabeads (Life technologies) for 45 min at 4C. Contaminating DNA was removed by treating the beads with Turbo DNase (2U in 20 μl). Crosslinked RNA was labeled by successive incubation with 5U Antarctic phosphatase (New England Biolabs) and 5U T4 PNK (New England Biolabs) in presence of 10 μCi [γ-32P] ATP (PerkinElmer, MA). Labeled material was resolved on 8% bis-tris gels, transferred to nitrocellulose membranes and exposed to autoradiography films for 4 hours. 100 pmol of a 3’-blocked DNA adapter were ligated to the RNA after dephosphorylation and before 5’ labeling by incubating the beads with T4 RNA ligase 1 (New England Biolabs) for 1 hour at 25C. After autoradiography, bands of interest were excised and the RNA eluted from the membrane by treating with proteinase K for 30’ at 37C and then proteinase K in presence of 3.5M urea for 30’ at 55C. Custom designed 5’ RNA adapters were ligated, the products size-selected on polyacrylamide gels.