title: GSM2123097: Cytoplasm-Total; Mus musculus; RNA-Seq
library_construction_protocol: We first lysed 5 million cells in 200 µl cold cell lysis buffer (10 mM Tris-HCl pH 7.5, 0.05% IGEPAL CA-630, 150 mM NaCl), incubating on ice for 5 minutes. We layered the cell lysate over 2.5 volumes of chilled sucrose cushion (24% sucrose in cell lysis buffer) and centrifuged at 15,000 × g for 10 minutes. The supernatant from this spin became the cytoplasmic fraction. After washing the pellet of nuclei with PBS (pH 7.5) + 1 mM EDTA, we resuspended the pellet in 100 µl of cold glycerol buffer (20 mM Tris-HCl pH 7.5, 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, 0.125 mM PMSF, 50% glycerol) by gently flicking the tube. We added 100 µl of cold nuclei lysis buffer (10 mM HEPES pH 7.5, 1 mM DTT, 7.5 MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M urea, 1% IGEPAL CA-630), then vortexed for four seconds. After 2 minutes on ice, we spun the nuclear lysate at 15,000 × g for 2 minutes. This supernatant was collected as the soluble nuclear (nucleoplasm) fraction. We rinsed the remaining pellet (chromatin fraction) in PBS + 1 mM EDTA, then resuspended the chromatin in 300 µl chromatin DNase buffer (20 mM Tris-HCl pH 7.5, 50 mM KCl, 4 mM MgCl2, 0.5 mM CaCl2, 2 mM TCEP, 0.5 mM PMSF, 0.4% sodium deoxycholate, 1% IGEPAL CA-630, 0.1% N-lauroylsarcosine) plus 15 µl murine RNase inhibitor (NEB) and 30 µl TURBO DNase (Ambion). The DNase digestion proceeded for 20 minutes at 37°C and was halted by adding 10 mM EDTA and 5 mM EGTA. Protein was digested with proteinase K for 1 hour at 37°C. RNA was isolated using Zymo RNA Concentrator-25 columns (two columns for the cytoplasmic fraction). From each cellular fraction, we sequenced total RNA and polyadenylated RNA (selected using oligo d(T)25 magnetic beads, NEB) using a strand-specific RNA-sequencing protocol for Illumina instruments described in Engreitz et al. Cell 2014.
attributes.tag: GEO Accession
attributes.value: GSM2123097