GSM2693605
GSM2693605: HeLa3; Homo sapiens; miRNA-Seq
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. 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 w2t = 2500 (w is in krpm; t is in hr)].  The gradients were calibrated using tobacco mosaic virus as a 200S sedimentation marker. Supraspliceosome peak fractions were confirmed by Western Blot (WB) and by electron microscopy visualization. WB analyses were performed, using anti-hnRNP G, kindly provided by Prof. Stefan Stamm (University of Kentucky, Lexington), visualized with horseradish peroxidase conjugated to affinity-pure Goat anti-Rabbit IgG (H+L; Jackson Immunoreaserch) diluted 1:5000. MAb-104, directed against the phosphorylated epitopes of the SR proteins was used as previously described. RNA was extracted from supraspliceosomes prepared from HeLa cells. The integrity of the RNA was evaluated by an Agilent 2100 bioAnalyzer For small RNA library construction, ~1 µg of RNA was used. After phosphatase and PNK treatments, the RNA was ethanol precipitated to enrich for small RNA, and small RNA libraries (in triplicates) were prepared according to NEBNext Small RNA Library Prep Set for Illumina (Multiplex Compatible) Library Preparation Manual. Adaptors were then ligated to the 5’ and 3’ ends of the RNA, and cDNA was prepared from the ligated RNA and amplified to prepare the sequencing library. The amplified sequences were purified on E-Gel® EX 4% Agarose gels (ThermoFisher # G401004), and sequences representing RNA smaller than 200 nt were extracted from the gel. The library was sequenced using the Illumina NextSeq 500 Analyzer.
GEO
GSM2693605
GEO Accession
GSM2693605