RiboSeq of human: fibroblasts: Sample S2.Riboseq2.7
Ribosomal profiling was performed using cycloheximide, following Ingolia et al. 2012 (Nature Protocols). The basic profiling protocol comprises cell or tissue lysis, nuclease footprinting, ribosome purification, and sequencing library generation and validation. Cells or tissues are rapidly lysed with detergent and the lysates are digested with E. coli RNase I in order to degrade mRNA not protected by the ribosome. Ribosomes, containing protected mRNA footprints, are purified by sedimentation through a sucrose cushion during ultracentrifugation. mRNA is recovered by gel size-selection from the ribosomal pellet and used to generate a sequencing library using an approach optimized for low sequence bias. For which we usually use small RNA Tru-seq kit from Illumina.