GSM6787994: ChrRNA_T2; Homo sapiens; OTHER
HeLa S3 cells from T0, T1, T2 and their cytoplasmic, nucleoplasmic and chromatin-associated RNA fractions were harvested. Two independent replicates for each time point were performed.  For that, cell pellets were lysed in 400 μl of cold cytoplasmic lysis buffer (0.15% NP-40, 10 mM Tris pH 7.5, 150 mM NaCl) and incubated on ice for 5 min. The lysates were layered onto 1 ml cold sucrose buffer (10 mM Tris pH 7.5, 150 mM NaCl, 24% sucrose w/v), and centrifuged at 3500 g for 10 min. Supernatant from lysates was centrifuged again at 14,000 x g for 1 min and its supernatant frozen as cytoplasmatic RNA fraction. Nuclear pellets from lysates were resuspended into 250 μl cold glycerol buffer (20 mM Tris pH 7.9, 75 mM NaCl, 0.5 mM EDTA, 50% glycerol). Then, 250 μl of cold nuclei lysis buffer (20 mM HEPES pH 7.6, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M urea, 1% NP-40, 1 mM DTT) was added to the samples, followed by a pulsed vortexing and incubated on ice for 2 min. Samples were then centrifuged for 2 min at 13,000 × g. The supernatant was frozen as nucleoplasmic RNA fraction and the pellet, representing the chromatin-associated RNA fraction, was rinsed with 1 ml of cold PBS and resuspended in 50 μl of PBS-EDTA. All buffer were suplemented with RNase inhibitors (RNasinPlus, Promega). Finally, only chromatin-associated RNA was extracted using TRIZOL and 5 μg of RNA was treated with TURBO DNA-free kit for DNA removal according to manufacturer's instructions. Libraries preparation of chromatin-associated RNA were realized using Illumina Stranded Total RNA prep ligation with Ribo-Zero plus kit, according to its reference guide.