Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: The cells of a 15 cm culture plate were lysed with lysis buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 200 mM KCl, 1 % Triton, 2 mM DTT, 100 µg/ml Cyclohexamide, 25 U/ml DNase and EDTA-free protease inhibitors, Roche). The mRNA not protected by ribosomes was digested with RNAseI and the ribosome footprints were collected by centrifugation with a continuous sucrose gradient (10-50 %) with DTT, cyclohexamideand 20 U/ml SUPERaseIN. The fraction with the ribosome footprint RNA was used for acid phenol extraction. Precipitated RNA was resuspended in 10 mM Tris pH7, mixed with 2x sample buffer (TBE-urea, NOVEX, Invitrogen) and loaded on a 15 % TBE-urea gel. The region between 26-34 nt was excised and used for library generation exactly as described previously (Ingolia et al, Nat Protoc 2012). The library was sequenced on the Illumina HiSeq system according to the manufacturer's protocol.