title: GSM1634443: C.rp.rep1; Homo sapiens; RNA-Seq
library_construction_protocol: Cells were treated with cycloheximide (100 μg/ml) for 5 minutes, washed with ice-cold phosphate-buffered saline (cycloheximide, 100 μg/ml), pelleted, and lysed in buffer A (20 mM Tris-HCl, pH 7.8, 100 mM KCl, 10 mM MgCl2, 1% Triton X-100, 2 mM DTT, 100 μg/ml cycloheximide, 1X complete protease inhibitor). Lysates were centrifuged at 5,000 rpm and the supernatant was treated with 2 U/μl of RNase I (Ambion) for 40 min at room temperature. Lysates were fractionated on a linear sucrose gradient (7% to 47%) using the SW-41Ti rotor at 36,000 rpm for 2 h. Fractions enriched in monosomes were pooled and treated with proteinase K (Roche) in a 1% SDS solution. Released RNA fragments were purified using TRIsure reagent and precipitated in the presence of glycogen. For libraries preparation, RNA was gel-purified on a denaturing 10% polyacrylamide urea (7 M) gel. A section corresponding to 30 to 33 nucleotides, the region where most of the ribosome-protected fragments are comprised, was excised, eluted and ethanol precipitated. The resulting fragments were 3′-dephosphorylated using T4 polynucleotide kinase (New England BioLabs, Inc.) for 6 h at 37°C in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (100 mM MES-NaOH, pH 5.5, 10 mM MgCl2, 10 mM β-mercaptoethanol, 300 mM NaCl). 3′ adaptor was added with T4 RNA ligase 1 (New England BioLabs, Inc.) for 2.5 h at 37°C. Ligation products were 5′-phosphorylated with T4 polynucleotide kinase for 30 min at 37°C. 5′ adaptor was added with T4 RNA ligase 1 for 18 h at 22°C. 3′ adaptor was added with T4 RNA ligase 1 (New England BioLabs, Inc.) for 2.5 h at 37°C. Ligation products were 5′-phosphorylated with T4 polynucleotide kinase for 30 min at 37°C. 5′ adaptor was added with T4 RNA ligase 1 for 18 h at 22°C.
attributes.tag: GEO Accession
attributes.value: GSM1634443