Ribo-seq of human: hepatocytes
Hepatocytes were grown to 90% confluence in a 10 cm culture dish and lysed in 1 ml cold lysis buffer (formulation as in TruSeq Ribo Profile Mammalian Kit,  RPHMR12126, Illumina) supplemented with 0.1 mg/ml cycloheximide. Homogenized and cleared lysates were then footprinted with Truseq Nuclease (Illumina) according to the manufacturers instructions. Ribosomes were purified using Illustra Sephacryl S400 columns (GE Healthcare), and the protected RNA fragments were extracted with a standard phenol:chloroform:isoamylalcohol technique. Following ribosomal RNA removal (Mammalian RiboZero Magnetic Gold, Illumina), sequencing libraries were then prepared out of the footprinted RNA by using TruSeq Ribo Profile Mammalian Kit according to the manufacturers protocol.