GSM2719992: M81-transformed LCLs sample 2; Homo sapiens; RNA-Seq
Lysates were clarified by centrifugation and ribosome covered RNA was isolated by sucrose gradient ultracentrifugation and subsequent fractionation. Isolated fractions were RNase I treated for 15 minutes at room temperature. Following nuclease treatment, ribosome protected fragments were gel purified. For total RNA sequencing, cells were lysed with Trizol (Ambion) reagent and RNA was purified according to manufacturer's instructions. Ribosome profiling libraries for high-throughput sequencing were generated according to the published protocol from Ingolia et al. 2012 published in Nature Protocols. The total RNA sequencing libaries were prepared by the DKFZ core facility using the strand-specific RNA preparation kit from Agilent.