Name: mouse_liver_ribo_1_s
Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: We generated Ribo-seq and matched RNA-seq data for the following samples: brain (cerebrum), liver, and testis samples from human (Homo sapiens), rhesus macaque (Macaca mulatta), mouse (Mus musculus, strain: CD-1, RjOrl:SWISS), grey short-tailed opossum (Monodelphis domestica), platypus (Ornithorhynchus anatinus), and chicken (red jungle fowl, Gallus gallus). Our study complies with all relevant ethical regulations with respect to both human samples and samples for the other mammals. Human samples were obtained from official scientific tissue banks or dedicated companies; informed consent was obtained by these sources from donors prior to death or from next-of-kin. The use of all human samples for the type of work described in this study was approved by an Ethics Screening panel from the European Research Council (ERC) (associated with H.K.'s ERC Consolidator Grant 615253, OntoTransEvol) and local ethics committees; that is, from the Cantonal Ethics Commission Lausanne (authorization 504/12) and Ethics Commission from the Medical Faculty of Heidelberg University (authorization S-220/2017). The use of all other mammalian samples for the type of work in this study was approved by ERC Ethics Screening panels (ERC Starting Grant 242597, SexGenTransEvolution, and ERC Consolidator Grant 615253, OntoTransEvol). The Ingolia 2012 protocol has been implemented in the TruSeq Ribo Profile kit (Illumina), which was used in our study. Specifically, frozen tissues were treated in 3 volumes of ice-cold lysis buffer (150 mM NaCl, 20 mM Tris-HCl pH7.4, 5 mM MgCl2, 5 mM DTT, 100 μg/ml cycloheximide, 1% Triton X-100, 0.5% Sodium deoxycholate, complete EDTA-free protease inhibitors (Roche) and 40 U/ml RNasin plus (Promega)) using a Teflon homogenizer. Lysates were incubated for 10 min on ice and cleared by centrifugation at 3,000 x g, 4°C for 3 min. Supernatants were flash-frozen and stored in liquid nitrogen. For absorbance measurements, lysates were gently thawed on ice and the OD260 was determined using a Nanodrop spectrophotometer (Thermo Fisher Scientific). From the lysate pool, 15 OD260 were incubated with 650 U RNase I (Ambion) and 5 U Turbo DNase (Ambion) for 45 min at room temperature and gentle agitation. Nuclease digestion was stopped through addition of 8.7 μl SUPERase In RNase Inhibitor (Ambion). Subsequently, lysates were applied to Sephacryl MicroSpin S-400 HR columns (GE Healthcare Life Sciences), pre-washed 3 times with 700 μl polysome buffer (150 mM NaCl, 20 mM Tris-HCl pH7.4, 5 mM MgCl2, 5 mM DTT, 100 μg/ml cycloheximide, complete EDTA-free protease inhibitors (Roche)) for 1 min at 600 x g, and centrifuged for 2 min at 600 x g and 4°C. The flowthrough was immediately mixed with 1 ml Qiazol (Qiagen) and ribosome-protected mRNA fragments were purified using the miRNeasy Micro kit (Qiagen) according to the manufacturer's instructions, and the concentration of the RNA was determined using a NanoDrop spectrophotometer. Prior to library preparation, a total of 5 μg RNA from each sample was subjected to ribosomal RNA depletion (Ribo-Zero rRNA Removal kit, Illumina) and subsequently purified using the RNA Clean & Concentrator-5 kit (Zymo Research) according to the manufacturer's protocol. The rRNA-depleted RNA was separated on a denaturing 15% urea polyacrylamide gel (Thermo Fisher Scientific) and stained with SYBR-Gold (Thermo Fisher Scientific). Gel slices between 26-34 nt were excised and the RNA was extracted using a 450 μl gel extraction buffer (0.5 M Ammonium acetate and 0.05% SDS) for 2 hours at room temperature and with gentle agitation. Gel pieces were removed by centrifugation over Spin-X filter tubes (Corning) for 2 min at 15,000 x g. RNA was precipitated over night at -20°C in the presence of 1 ml 100% ethanol and 3 μl glycogen. RNA was pelleted for 25 min and washed with 80% ethanol in a tabletop centrifuge at maximum speed and 4°C. Sequencing libraries were generated using the TruSeq Ribo Profile Library Prep Kit (Illumina). End-repair, 3' adapter, reverse transcription, cDNA purification, and circularization were done according to the manufacturer's instructions. For opossum, platypus, and chicken samples, an additional rRNA depletion step was implemented, given that the standard depletion step is based on human, mouse and rate sequence information and was determined to be inefficient for these non-model species (which are evolutionarily highly diverged from human/mouse) in test experiments. Specifically, to reduce rRNA contamination for these species (and thus reduce the number of sequencing reads needed for in-depth analyses), first strand cDNAs derived from species specific rRNA contaminants were further depleted after circularization by hybridization to 5'-biotinylated sense strand oligonucleotides followed by removal of the duplexes through streptavidin affinity as previously described. PCR amplification of the circularized cDNA product was done using the TruSeq Ribo Profile Library Prep Kit (Illumina) according to the manufacturer's instructions. The final library of 150-200 bp was gel-purified on a 10% polyacrylamide non-denaturing gel (Thermo Fisher Scientific), excised and recovered with 330 μl gel extraction buffer for 1 hour at 37°C and gentle agitation. Gel pieces were removed by centrifugation over Spin-X filter tubes (Corning) for 2 min at 15,000 x g. Libraries were precipitated at -20°C for 1 hour in the presence of 525 μl 100% isopropanol and 2 μl glycogen, pelleted for 25 min at 4°C and 15,000 x g, washed with 80% ethanol and resuspended in water. In parallel to Ribo-seq library preparation, matched RNA-seq libraries were prepared from the same lysates based on Ingolia 2012 and using TruSeq Ribo Profile Library Prep Kit. The concentration and the quality of both the Ribo-seq and RNA-seq libraries were determined using Qubit (Thermo Fisher Scientific) and Fragment Analyzer (Advanced Analyticals) platforms.