title: GSM3326048: WT_TOT_Rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Once the 80% confluence was reached, cells were incubated for 3–4 min with 10 µg/ml cycloheximide at 37°C  to block translational elongation. Cells were washed with PBS + 10 µg/ml cycloheximide and scraped directly on the plate with 300 µl lysis buffer (10 mM NaCl, 10 mM MgCl2, 10 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1% sodium deoxycholate, 0.2 U/µl RNase inhibitor [Fermentas  Burlington, CA], 10 µg/ml cycloheximide, 5 U/mL Dnase I [New England Biolabs, Hitchin, UK] and 1 mM DTT) and transferred to a tube. Nuclei and cellular debris were removed by centrifugation for 5 min at 13,000 g at 4°C. The polysome-containing lysate was stored at -80 °C or directly layered onto a linear sucrose gradient (15–50% sucrose (w/v), in 30 mM Tris–HCl at pH 7.5, 100 mM NaCl, 10 mM MgCl2) and centrifuged in a SW41Ti rotor (Beckman Coulter, Indianapolis, IN) at 4 °C for 100 min at 180,000 g. Ultracentrifugation allows the polysome separation according to the sedimentation coefficient of macromolecules. After ultracentrifugation, gradients can be fractionated and mRNAs in active translation, corresponding to polysome containing fractions, separated from untranslated mRNAs. Fractions of 1 mL volume were collected with continuous monitoring absorbance at 254 nm. Total RNA was obtained by pooling together a fixed amount (20%) of  each collected fraction. To extract RNA,  polysomal and total fractions were  treated directly with 0.1 mg/ml proteinase K (Euroclone,  Italy) for 2 h at 37°C. After phenol–chloroform extraction and isopropanol precipitation, polysomal RNA was resuspended in 30 µl of RNAse-free water. Both total RNA and polysome RNA were analyzed on an Agilent Bioanalyzer to assess RNA integrity and quantified using the QBit. RNA libraries were prepared for sequencing using standard Illumina protocols: the Illumina TruSeq Targeted RNA Custom Panel Kit was used with 500 ng of total or polysomal RNA.
attributes.tag: GEO Accession
attributes.value: GSM3326048