GSM5066052: RA_Polysome3; Homo sapiens; RNA-Seq
RNA purification from RNaseI footprinted samples was performed by Trizol RNA extraction. RNA purification from polysome fractions  was  performed  by  isopropanol  precipitation,  followed  by  TURBO  DNase  treatment, acidic phenol/chloroform RNA purification and ethanol precipitation at -80oC overnight. RNA concentration was determined by Nano-drop 2000 software. Two rounds of polyA selection from polysome fractions were performed using oligo (dT) Dynabeads PolyA RNA  was  fragmented  by  alkaline  hydrolysis. 28–34  nt  ribosome  footprints  and  50–80  nt  mRNA fragments were gel purified in 10% (w/v) polyacrylamide-TBE-Urea gel. Ribosome footprints were subjected to rRNA depletion (Illumina RiboZero rRNA removal kit). 5' stranded libraries were constructed using NEB Next Multiplex Small RNA Library Prep. Resulting cDNA was PCR amplified and gel purified prior to sequencing. Libraries were subjected to 75bp single end RNA Sequsing NextSeq 500 Illumina sequencer,High OutputKit v2.5 (75 Cycles)(Next Generation Sequencing Facility, Faculty of Medicine, University of Leeds). RNA-seq, Ribo-seq