Nuclear Roles of Spliceosome-Associated microRNAs in Neuronal Cancer Cells
Nuclear Roles of Spliceosome-Associated microRNAs in Neuronal Cancer Cells
Supraspliceosomes were prepared, as previously described {https://www.sciencedirect.com/science/article/pii/S1097276504004459?via%3Dihub }, from nuclear supernatants enriched in supraspliceosomes from the following cell- lines: HCN, U87MG and SH-SY5Y. Briefly, nuclear supernatants were prepared from purified cell nuclei by microsonication of the nuclei and precipitation of the chromatin in the presence of excess of tRNAs. All isolation steps were conducted at 4°C. The nuclear supernatant was fractionated on 10-45% (vol/vol) glycerol gradients. Centrifugations were carried out at 4°C in an SW41 rotor run at 41 krpm for 90 min [or an equivalent 2t = 2500 ( is in krpm; t is in hr)]. The gradients were calibrated using the tobacco mosaic virus as a 200S sedimentation marker. Supraspliceosome peak fractions were confirmed by Western Blot (WB) and by electron microscopy visualization. RNA extraction NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (Set 1)