title: GSM2367484: PAR-CLIP DDX54 wild-type actinomycin D treated; Homo sapiens; RIP-Seq
library_construction_protocol: Cell pellets were lysed in approximately 3 cell pellet volumes (5 ml) of high salt NP-40 lysis buffer (50 mM Tris pH 7.5, 500 mM NaCl, 1% (v/v) NP-40, 1 mM DTT, complete EDTA-free protease inhibitor cocktail (Roche)), incubated for 30 minutes on ice followed by 10-second sonication at 80% amplitude. Immunoprecipitation (IP), small RNA cloning and cDNA library generation were performed as previously described (Hafner et al., 2010; Hafner et al., 2012) with the following minor modifications. Briefly, the first and second RNase T1 treatments were performed at 1 U/µl for 10 min at 22 °C and at 2 U/µl in 80 µl of IP wash buffer (50 mM HEPES, pH 7.5, 300 mM KCl, 0.05% (v/v) IGEPAL CA-630, 0.5 mM DTT) for 7 min at 22 °C, respectively. Crosslinked protein-RNA complexes were resolved by SDS-PAGE using Novex 3-8% gradient Tris-acetate gels (Thermo Fischer). Proteins and protein-RNA complexes were transferred to a nitrocellulose membrane (Whatman) and exposed to phosphorimager screen. 32P-RNA-FLAG/HA-DDX54 complex migrating at approximately 115 kDa was excised and proteinase K (Roche) digested (4 mg/ml for 1 h at 55 °C).  After phenol-chloroform extraction and precipitation, RNA was ligated to 3’ adaptor (see Oligonucleotides) and gel purified. Two RNA size ranges per each sample (corresponding to 22-35 and 50-100 nucleotides of unligated fragments) were excised, 5’adaptor ligated, reverse transcribed and PCR-amplified to obtain cDNA libraries. cDNA libraries were amplified using Phusion High-Fidelity DNA polymerase (Thermo Fischer) and different barcode containing reverse PCR primers (see Oligonucleotides) followed by gel purification using QIAquick gel extraction kit (Qiagen). Clustering and amplification of cDNA libraries was performed according to manufacturer's instruction. Massively parallel sequencing was carried out on HiSeq2000 Illumina instrument using 1x51+7 cycle and 1x101+7 cycle runs. small RNA cloning (Hafner et al, 2012)