title: GSM2480997: NSC11 cells mock irradiated, collected at 1h, Total RNA, biological replicate 1; Homo sapiens; RNA-Seq
library_construction_protocol: For polysome-bound RNA, cells incubated in 100 μg of cycloheximide/mL for 15 minutes before collection. Cytoplasmic RNA was obtained by lysing cells in 1 mL of polysome buffer [15 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, 15 mmol/L MgCl2, 1% Triton X100, 100 μg/mL cycloheximide, 1 mg/mL heparin, and 500 units/mL RNasin (Promega)]. After 15 minutes on ice, lysates were centrifuged (12,000 × g for 15 minutes), and the resulting cytosolic supernatant was layered onto a 10% to 50% sucrose gradient. Gradients were then centrifuged at 35,000 × g for 3 hours at 4°C and polysome-bound fractions were collected using an ISCO Density Gradient Fractionation System (ISCO, Lincoln, NE) with continuous monitoring based on A254. The RNA in each fraction was extracted using TRIzol LS (Invitrogen).  Trizol extraction of total RNA was performed according to the manufacturer's instructions. between 100ng to 1µg of total RNA was used as the input for mRNA capture with oligo-dT coated magnetic beads.  The mRNA was fragmented followed by random-primed cDNA synthesis. The resulting double-strand cDNA was used as the input to a standard Illumina library prep with end-repair, adapter ligation and PCR amplification to generate a sequencing ready library.  The final library was then quantitated by qPCR before cluster generation and sequencing on the Illumina HiSeq2500 sequencer.
identifiers.namespace: GEO
identifiers.id: GSM2480997