title: GSM4677993: PAR-CLIP p300 ESC rep2; Mus musculus; RIP-Seq
library_construction_protocol: For PAR-CLIP, approximately 200x106 ES cells, were acutely treated for 16 h with 100 μM 4-thioridine (4-SU) (Sigma) and were cross-linked using 150 mJ/cm2 of 365 nm UV light in a UVP crosslinker (Analytic Jena). The isolated nuclei pellets were lysed on ice with NP40 Lysis buffer (150 mM KCl, 50 mM HEPES pH 7.5, 2 mM EDTA, 1 mM NaF, 0,5% NP40, 0.5 mM DTT, protease inhibitors). The cleared lysates were subjected to SUPERase-In digestion using RNAase A/T1 cocktail enzyme mix (Ambion) for 3 min at 37°C to improve IP efficiency. The cross-linked p300-RNA complexes are immunoprecipitated with p300 antibody (Millipore) bound to BSA blocked protein G-Dynabeads (Life technologies). Beads were washed 3 times with IP wash buffer (300 mM KCl, 50 mM HEPES, pH 7.5, 0,05% NP40, 0.5 mM DTT, SUPERase-In, protease inhibitors) and  resuspended in dephosphorylation buffer by adding Recombinant Shrimp Alkaline Phosphatase (rSAP, NEB) for 15 min at 37°C to dephosphorylate the RNA. The beads were washed with Phosphatase wash buffer (50 mM Tris-HCl, pH 7.5, 20 mM EGTA, 0.5% NP40, SUPERase-In, protease inhibitors) and T4-PNK buffer (50 mM NaCl, 50 mM Tris HCl pH 7.5, 10 mM MgCl2, 5 mM DTT, SUPERase-In, protease inhibitors) and were incubated with non-radioactive ATP. The protein-RNA complexes were separated by Novex Bis-Tris 4–12% polyacrylamide gel (Invitrogen) for 55 min at 220 V. p300-RNA bands that correspond to the expected size of p300 revealed in Input were excised from the nitrocellulose membrane, eluted by incubating with RNA elution buffer (50 mM NaCl, 50 mM HEPES pH7.0, 1 mM EDTA, 2 mM CaCl2, 1% SDS, SUPERase-In) and digested with Proteinase K (4mg/ml, NEB) and 3.5M Urea for 30 min at 55°C. RNA was purified using acidic phenol/chloroform (ThermoFisher) followed by ethanol precipitation. PAR-CLIP libraries were prepared using NEB next small RNA library set for Illumina sequencing (NEB), according to manufacturers instructions and were checked by Bioanalyzer (Agilent) for quality. pproximately 2 μg of total RNA were subjected to poly(A) selection, and libraries were prepared using the TruSeq RNA Sample Prep Kit (Illumina) following manufacturer's instructions. Sequencing was performed on the Illumina NextSeq 500 platform.
attributes.tag: GEO Accession
attributes.value: GSM4677993