GSM1963691: Gal4-DBD background control (Replicate 1); Mus musculus; OTHER
CBP/p300 flox/flox MEFs or background control strains were incubated for 16h with 150uM 4-thiouridine (4-SU). Plates were washed with PBS before cells were cross-linked using 150mJ/cm2 365nM UV light with a stratalinker UV crosslinker (Stratagene). All subsequent stages of PAR-CLIP were carried out immediately to prevent degradation of cross-linked CBP. Cells were harvested by scraping in  PBS + 1mM EDTA and lysed by incubating for 10 minutes on ice with CLIP lysis buffer A (20mM Hepes KOH pH7.9, 150mM KCl, 5mM EDTA, 2% EMPIGEN, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, 0.1% SDS, 2mM DTT) supplemented with Complete EDTA free protease inhibitor (Roche), Murine RNase inhibitor (NEB), Turbo DNase I (Ambion) and sonicated for 5x 30s cycles in a bioruptor (Diagenode). Cell lysate was cleared by centrifugation at 20000xg for 30 minutes. The cleared cell lysates were subjected to an initial partial RNAse digestion step using RNase/T1 cocktail (4000x) (Ambion) for 5 min at 21C to improve immunoprecipitation efficiency. Immunopreciptations were carried out using a depleting amount of anti-CBP antibody (Santa Cruz, sc-583) or anti-Flag M2 (Sigma, F1804) for 2 hours at 4C. Lysates were then subjected to a second, more stringent RNAse treatment using RNase A/T1 cocktail (10x) for 15min at 37C before recovering immunocomplexes for 30 minutes with BSA blocked Protein A dynabeads (Life Technologies). The digestion with higher RNase concentration was important for optimising transfer efficiency of the large (>250KDa) CBP-RNA complexes.  Beads were collected and washed twice with CLIP wash buffer (20mM Tris HCl pH 7.9 (4C), 400mM KCl, 5mM EDTA, 0.5mM EGTA, 2% EMPIGEN, 0.7% Na-deoxycholate, 0.5% N-lauroylsarcosine, 2M Urea, 2mM DTT, protease inhibitors) then IP50 buffer (20mM Tris HCl pH 7.9 (4C), 50mM KCl, 0.2mM EDTA, 10% Glycerol, 2mM DTT), before resuspending in 1x Turbo DNase buffer. CLIP wash buffer contains 2M Urea, which was important for removing non-specific RNA binding whilst retaining immunoprecipitated protein-RNA complexes. Contaminating DNA was removed by incubation with 2.5U Turbo DNase I for 30 minutes at 37C. Beads were washed with CLIP wash buffer and IP50 buffer. Cross-linked RNA complexes were dephosphorylated by incubating with Recombinant Shrimp Alkaline Phosphatase (rSAP, NEB) for 15 minutes at 37C, washing with Phosphatase wash buffer (20mM Tris HCl pH 7.9 (4C), 400mM KCl, 10mM EDTA, 10mM EGTA, 0.05% IGEPAL CA-630, 2mM DTT) then T4-PNK buffer (50mM Tris HCl pH 7.5 (RT), 50mM NaCl, 10mM MgCl2, 5mM DTT). Protein-RNA complexes were then labelled by incubating with 5U T4 PNK (NEB) with 10uCi g-32P-ATP for 15 minutes at 37C. Labelled material was washed again with CLIP wash buffer and IP50 before denaturing with LDS loading buffer and resolving on a 3-8% Tris-acetate gel. Gels were transferred to 0.2uM PVDF membrane for 12h at 40V, then exposed to autoradiography screens or x-ray film for 12-24h to image labelled RNA complexes. For sequencing experiments, labelled CBP-RNA bands (or control bands) were excised from the membrane and eluted by incubating with RNA elution buffer (Tris HCl pH8.0 (RT), 50mM NaCl, 1mM EDTA, 2mM CaCl2, 1% SDS) and Proteinase K (4mg/ml, Roche) for 30 minutes at 37C, then for 30 minutes at 55C with RNA elution buffer + 3.5M Urea and Proteinase K. RNA was then purified using acidic phenol:chloroform (Ambion) and ethanol precipitation. Sequencing libraries for PAR-CLIP experiments were prepared using NEB next small RNA library set for Illumina sequencing (New England Biolabs) according to manufacturers instructions. Libraries were checked for quality by bioanalyzer before and after size selection by Pippin prep and were 50bp single-end sequenced on an Illumina NextSeq 500.
GEO Accession
GSM1963691