GSM8767465: Liver Ercc1 d/- 30% DR 16wks #4; Mus musculus; OTHER
Snap frozen tissue samples from the same livers as used for mRNA sequencing were used to perform ribosome profiling with minimal modifications as previously described. Samples were powdered with a BioSpec Pulverizer (#59012MS) on dry ice. Pulverized material was lysed with 500ul ice-cold lysis buffer (20 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 0.1% IGEPAL CA-630 (Sigma-Aldrich, USA), 1 mM DTT, 10 U/ml RNase-free DNase 1 (Lucigen, USA), 100 µg/ml cycloheximide (Sigma-Aldrich, USA)) for 15 minutes on ice, triturated ten times through a 23G needle, and clarified through centrifugation at 20000 g at 4 °C for 10 min. Total RNA content of sample lysates was estimated using the Qubit™ RNA broad range (BR) Assay Kit (Thermo Fisher Scientific, USA) on an Invitrogen™ Qubit™ 4 fluorometer (Thermo Fisher Scientific, USA). Lysates were then digested per 200 µl aliquots with RNAse I (10 units per 20 µg of RNA; Lucigen, USA) for generation of ribosome-protected fragments (RPFs) at 20 °C for 45 min while shaking at 400 RPM on a thermomixer. The digestion reaction was stopped by adding 5 µl (5U) of SUPERase*In RNAse inhibitor (Thermo Fisher Scientific, USA) and placing the samples on ice. Digested lysates were transferred to Microspin S-400 HR sephacryl columns (Sigma-Aldrich, USA) equilibrated with 3 ml of cold RNase-free polysome buffer (20 mM Tris-Cl pH 7.4, 150 mM NaCl, 5 mM MgCl2) and centrifuged at 600g at room temperature for 2 min. 10 µl 10% SDS was added to the digested lysates and RPFs were extracted using 3 volumes (660 µl) of Trizol LS (Fisher Scientific, USA), followed by addition of 1 volume (880 µl) of ethanol and RNA purification using the Zymo Direct-zol RNA micro prep kit (Zymo Research, USA) with slight modifications to the manufacturer's instructions: Columns were spun dry for 1 min at 12000 g and isolated RPFs were eluted in 20 µl nuclease-free water (Sigma-Aldrich, USA). Depletion of rRNA was performed using RiboPOOL technology (siTOOLs Biotech; riboPOOL ribo-seq cat# dp-K012-000052, Germany) with a slight modification to the manufacturer's instructions: 200 pmol of RiboPOOL and 100 µl of beads were used per sample. Next, RNA was purified using the Zymo RNA Clean and Concentrator-5 kit (Zymo Research, USA) and size-selected through denaturing PAGE using 15% TBE-Urea gels (Thermo Fisher Scientific, USA). RNA fragments corresponding to 26–34 nucleotides were excised and recovered from gel slices by crushing the gel and rocking at 20 °C, 700 RPM for 2 hours on a thermomixer. RNA solutions were transferred to Costar Spin-X filter tubes (Thermo Fisher Scientific, USA) and filtered through centrifugation at 2350 g for 6 min. 2 µl of GlycoBlue (Thermo Fisher Scientific, USA) and 700 µl of isopropanol were added per aliquot and RNA was left to precipitate overnight at −80 °C. Following precipitation, RNA fragments were pelleted by centrifugation at 21130 g at 4 °C for 45 min. Pellets were washed with 1ml of 80% ice-cold ethanol and centrifuged at 21130 g at 4 °C for 10 min. RNA pellets were air-dried for 3-4 min and dissolved in 60.75 µl nuclease-free water on ice. RNA fragments were dephosphorylated using 30 U of T4 PNK (Lucigen, USA) for 1 h at 37 °C and purified using Zymo RNA Clean and Concentrator-5 kit (Zymo Research, USA), where isolated RNA fragments were eluted in 9.5 µl nuclease-free water. Purified fragments were ligated to a pre-adenylated 3'oligonucleotide linker using 100 U of T4 RNA ligase 2 Deletion Mutant (Lucigen, USA) and 5 U T4 RNA ligase 1 (Thermo Fisher Scientific, USA) at 23 °C for 3 h. Leftover linker was removed using 5'Deadenylase (New England Biolabs, USA) and Rec J Exonuclease (Lucigen, USA). Linker-ligated RNA fragments were reverse-transcribed into cDNA using EpiScript reverse transcriptase (Lucigen, USA). cDNA was treated with Exonuclease I (Lucigen, USA) for 30 min at 37 °C, followed by 15 min at 80 °C with a reduction to 4 °C; and further treated with 5 U of RNAse I (Lucigen, USA) and 2.5 U of Hybridase Thermostable RNase H (Lucigen, USA) at 55 °C for 5 min followed by an incubation step at 4 °C to stop the reaction. Treated cDNA was purified using the Zymo Oligo Clean and Concentrator Kit (Zymo Research, USA) with modifications to the manufacturer's instructions: Columns were spun dry for 1 min at 21130 g and isolated RPFs were eluted in 9.5 µl nuclease-free water (Sigma-Aldrich, USA).  Size-selection of cDNA containing ligated linkers was performed through denaturing PAGE using 10% TBE-Urea gels (Thermo Fisher Scientific, USA). cDNA fragments corresponding to 70–80 nucleotides were excised and extracted with ammonium acetate and SDS, followed by overnight precipitation with isopropanol as described above. Size-selected cDNA was circularized for 3 h at 60 °C using 100 U of circLigase I (Lucigen, USA) followed by heat inactivation for 10 min at 80 °C and amplified using Phusion high-fidelity polymerase (New England Biolabs, USA) with reverse primers containing unique barcode sequences for 10 cycles of: 30 sec at 98 °C, 15 sec at 94 °C, 5 sec at 55 °C, 10 sec at 65 °C. Following amplification, 5 µl of 3 M NaCl (Thermo Fisher Scientific, USA), 1 ml of ethanol and 2 µl of GlycoBlue (Thermo Fisher Scientific, USA) were added to each aliquot of cDNA and left to precipitate overnight at −80 °C as described above.  Amplified cDNA libraries were size-selected using 8% non-denaturing TBE-Urea gels (Thermo Fisher Scientific, USA). cDNA libraries corresponding to 150 nucleotides were excised and recovered from gel slices by crushing and subsequent rocking at 37 °C at 700 RPM on a thermomixer for 2 hours. cDNA solutions were transferred to Costar Spin-X filter tubes (Thermo Fisher Scientific, USA) and filtered through centrifugation at 2350g for 6 min. cDNA libraries were purified using the Zymo DNA Clean and Concentrator-5 kit (Zymo Research, USA) with modifications to the manufacturer's instructions: Columns were spun dry for 2 min at 12000 g and isolated RPFs were eluted in 13 µl nuclease-free water (Sigma-Aldrich, USA).  cDNA libraries were quantified using Qubit™ DNA high sensitivity (HS) Assay Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions on an Invitrogen™ Qubit™ 4 fluorometer (Thermo Fisher Scientific, USA) and Bioanalyzer 2100 (Agilent) using the High Sensitivity DNA kit and pooled in equimolar ratios. Sequencing was performed on a NextSeq2000 (Illumina; 1x50bp) at the Utrecht Sequencing Facility (USEQ) to a depth varying between 37831148 and 70879824 total reads per sample.
GEO
GSM8767465_r1