title: GSM8508335: Cycling Tg Treated RNA-seq, 2; Homo sapiens; RNA-Seq
library_construction_protocol: RNA was extracted using by drop-wise addition of polysome lysis buffer (20 mM Tris pH 7.4, 50 mM NaCl, 5 mM MgCl2, 1 mM DTT, 50 mM KCl, 1% Triton x-100, 0.1 mg/mL cyclohexamide, 1X HALT protease-phosphatase inhibitors). 50 ug of bulk RNA from lysate (measured by nanodrop) was used for Trizol purification of RNA-seq and 50 ug of lysate was prepared for Ribo-seq. Ribo-seq lysates were treated with 1 uL of RNAse I (thermofisher) and incubated at 4C with rotation for 40 min; reaction was then stopped with 100 U of SUPERasein (thermofisher). Lysates were then centrifuged through a sucrose cusion to precipitate ribosome pellets. Pellets weree then suspended in Trizol for RNA preparation. Prepared RNA was DNAse I treated and then gel purified for riboseq footprints (25-40 nts) followed by extraction. Extracted RNA was then depleted of rRNA (thermofisher). RNA-seq RNA was prepared using Tru-Seq Stranded RNA library kit (Illumina) while Ribo-seq RNA was prepared using NEBNext Multiplex Small RNA Library prep (New England Biolabs)