GSM5827702: reporter RNA from the subpolysomal fraction of HeLa cells treated with Torin1 for 2 hours, rep 1; Homo sapiens; RNA-Seq
Either total RNA was extracted directly with Trizol containing a capped RNA spike-in molecule, or cells were lysed on plate in 20 mM Tris pH 7.4; 150 mM NaCl; 5 mM MgCl2; 1% Triton X-100; 1 mM DTT; 100 ug/ml cycloheximide. Clarified lysates were then separated into a subpolysomal and polysomal fraction on a 5-50% sucrose gradient, and an equal amount of the capped spike-in RNA described above was added to each fraction. Total RNA was retrieved from both fractions by proteinase K treatment followed by acid phenol extraction. The Renilla luciferase RNA was reverse transcribed with a gene-specific primer, followed by alkaline RNA hydrolysis. The 3' end of the cDNA was then ligated to a double stranded DNA linker with a 3' NNNNNN or GNNNNN (mixed in 1:4 ratio). A 3' adapter sequence was added by PCR, followed by a second PCR that incorporates Illumina  sequences with dual index. Sequencing libraries from plasmid were prepared by adding adapter sequences in a first PCR step, followed by incorporation of Illumina  sequences in a second PCR reaction.