title: GSM2674847: WT Nucleoplasmic polyA RNA rep-3 run-1; Mus musculus; RNA-Seq
library_construction_protocol: Wild-type mouse ES cells from one confluent maxi dish were trypsinized and collected by centrifugation at 1200 rpm for 3 min in 15 ml falcon tubes. The cell pellet was resuspended in PBS and the cell number determined using a CASY-counter. 2x107 cells were resuspended in 200 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 lysate was layered onto 500 μl of cold sucrose buffer (10 mM Tris pH 7.5, 150 mM NaCl, 24% sucrose w/v), and centrifuged in microfuge tubes at 14,000 rpm for 10 min at 4°C. The supernatant recovered after this spin (~700 μl) represents the cytoplasmic fraction. The nuclear pellet was gently resuspended in 200 μl of cold glycerol buffer (20 mM Tris pH 7.9, 75 mM NaCl, 0.5 mM EDTA, 50% glycerol, 0.85 mM DTT. An additional 200 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 pulsed vortexing and incubation on ice for 1 min. Samples were then centrifuged in microfuge tubes for 2 min at 14,000 rpm at 4°C. The supernatant from this spin represents the soluble nuclear fraction (nucleoplasm), while the remaining insoluble part represents the chromatin pellet. TrueSeq total RNA TrueSeq mRNA
attributes.tag: GEO Accession
attributes.value: GSM2674847