title: GSM3763621: Left ventricle tissue of end-stage DCM patients (20RW01403); Homo sapiens; OTHER
library_construction_protocol: Briefly, snap-frozen cell pellets or 50-100 mg of tissue, previously powdered under liquid nitrogen, were lysed in 1ml cold lysis buffer (formulation as in TruSeq Ribosome Profile, Illumina) supplemented with 0.1mg/ml cycloheximide (CHX) to stabilize ribosomal subunits and prevent post-lysis translocation. Homogenized and cleared lysates were then footprinted with Truseq Nuclease (Illumina) according to manufacturer's instructions. Ribosomes were purified using Illustra Sephacryl S400 columns (GE Healthcare) and the protected RNA fragments were extracted with standard phenol:chloroform:isoamylalcohol technique. Following ribosomal RNA removal (Mammalian RiboZero Magnetic Gold, Illumina), sequencing libraries were prepared out of the footprinted RNA. Ribo-seq libraries were pooled to perform multiplex sequencing on Illumina Hiseq machines.