title: GSM3019386: ED170911; Mus musculus; RNA-Seq
library_construction_protocol: All samples: Cells were resuspended in TRIzol reagent, flash frozen, and stored overnight at -80°C. Cell lysates were chloroform extracted once and total RNA purified by isopropanol precipitation. Contaminating DNA was digested with Turbo DNase. Samples were purified by phenol:chloroform extraction followed by a second isopropanol precipitation and resuspension in RNase-free water. 1-5 μg RNA in 9 μL RNase-free water was mixed with 1.5 μL 10x binding buffer (1 M NaCl, 100 mM HEPES pH 7.4, 10 mM EDTA, 0.5% Tween), and 3 μL DMF, added to MTS resin (10 μL aliquot), and incubated at room temperature in the dark for 2 h with rotation. Beads were captured on a magnetic rack and supernatant removed. Beads were incubated for 5 min with the following wash buffers: once with 100 μL 1x binding buffer, twice with 100 μL high salt wash buffer (1M NaCl, 100 mM Tris pH 7.4, 10 mM EDTA, 0,05% Tween), twice with 100 μL denaturing buffer (8 M guanidinium chloride), and three times with 100 μL buffer TE (10 mM Tris pH 7.4, 1 mM EDTA) at 55°C. RNA was eluted with 10 μL elution buffer (100 mM NaCl, 10 mM Tris pH 7.4, 1 mM EDTA, 10 mM DTT, and 100 pg fragmented total RNA from S. pombe), and incubated at room temperature in the dark for 15 min with rotation. Eluent was ethanol precipitated with 5-10 μg glycoblue, and RNA concentration was assayed by Bioanalyzer RNA 6000 Pico Kit (Agilent) according to the manufacturer's instructions. For s4U Chase-Seq and TT-Seq: total RNA was sheared prior to s4U-RNA enrichment with MgCl2 at 94℃ for 4 min. 10 ng input and enriched RNA were used for rRNA-depleted library preparation. Sequencing libraries were constructed using the Clontech RNA Pico Input mammalian kit.
attributes.tag: GEO Accession
attributes.value: GSM3019386