GSM6016335: U251 cells 10nM (-)-SDS-1-021 treated, collected at 6h, polysomal RNA, biological replicate 1; Homo sapiens; RNA-Seq
Cells were incubated in 0.1 mg of cycloheximide/mL for 10 minutes before collection. Cytoplasmic RNA was obtained by lysing cells in 1.5 mL of polysome buffer [15 mmol/L Tris-HCl (pH 7.5), 300 mmol/L NaCl, 15 mmol/L MgCl2, 1% Triton X100, 0.1 mg/mL cycloheximide, and 1 mg/mL heparin]. After 10 minutes on ice, lysates were centrifuged (10,000 × g for 10 minutes), and the resulting cytosolic supernatant was layered onto a 10% to 50% sucrose gradient. Gradients were then centrifuged at 35,000 rpm for 3 hours at 4°C in an SW41Ti rotor 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).  Excess heparin was removed by two rounds of LiCl precipitation.  The resulting RNA was DNase digested using the on-column RNA clean up protocol with the QIAGEN RNeasy Mini Kit. 200ng 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 HiSeq4000 sequencer.