title: GSM1151219: Top3b; Homo sapiens; OTHER
library_construction_protocol: The RNA topoisomerase assay followed a previously published protocol. Briefly, the P-labled purified RNA circle substrate (1 nM) was incubated with the indicated topoisomerase in 10 μl of reaction buffer containing 20 mm Tris-HCll  (pH 7.5), 5 mm MgCl2, 2 mm DTT, 0.1 mg/ml BSA, 5% glycerol and 10% PEG400 at 37 °C for 90 min. Reactions were terr minated by adding 10 μl stop buffer (0.5% SDS, 20 mm EDTA and 0.2 mg/ml proteinase K) and incubated at 50 °C for    30 min. After phenol/chloroform extraction and ethanol precipitation, the mixtures were fractionated on 15% TBE-Urea gels and analyzed by autoradiography or PhosphoImager. To study whether Top3β binds mRNAs in vivo and also to identify its genome-wide RNA targets, we performed HITS-CLII P (high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation). In this method, proteins are covalently crosslinked with bound RNAs in vivo, thus allowing specific mapping of the sites of interaction of the protein with the RNA. The level of RNA obtained by Flag-Top3β IP from HeLa cells expressing HFTop3β was significantt ly higher than the background signal from a mock IP using cells that do not express the tagged protein, indicating that Top3β binds RNA in vivo. The majority of 2.4 million sequencing readhad a sense orientation, and the density   of tags within exons was 58-fold higher than within introns, suggesting that Top3β binds mRNA.