GSM2176668: input siRAM rep3; Mus musculus; RNA-Seq
Cells were incubated in 100 µg/ml cycloheximide for 10 min, washed in ice cold PBS supplemented with 100 µg/ml cycloheximide  and extracts were collected in polysome lysis buffer: 15 mM Tris (pH7.5), 15 mM MgCl2, 0.3 M NaCl, 1 mM DTT, 1% Triton X-100, 100 mg/ml cycloheximide, 100 U/ml RNasin. 10% of extracts was kept as input and the rest layered onto 10 ml of 10%–50% sucrose steps and centrifuged at 18 000 x g for 2 hr at 4ºC. 0.5 ml fractions were collected and OD254 nm monitored. Fractions 17-22 (polysome) were purified by Phenol:Chloroform:Isoamyl Alcohol (25:24:1) (Ambion), pooled, and RNA precipitated with 2M of LiCl (Sigma). RNA from inputs was purified using Trizol Reagent (Invitrogen) according to manufacturer’s instructions. Libraries were prepared according to Illumina's TruSeq Stranded Total RNA with Ribo-Zero Gold for Human/Mouse/Rat (RS-122-2303). Briefly, the first step involves the removal of ribosomal RNA (rRNA) using biotinylated, target-specific oligos combined with Ribo-Zero rRNA removal beads. The Ribo-Zero Gold Human/Mouse/Rat kit depletes samples of cytoplasmic rRNA and mitochondrial rRNA. Following purification, the RNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments are then 'A' tailed followed by ligation of the Illumina adapter. The products are purified and enriched by PCR to create the final cDNA library.
GEO Accession
GSM2176668