GSM9742568: Liver, ID, 2, Ribo_Seq; Mus musculus; OTHER
Mice liver tissue was snapfrozen after isolation. Tissues were homogenized in lysis buffer (20 mM Tris, pH 7.4, 140 mM KCl, 5 mM MgCl2, 1.0 % Triton X-100, 1 mg/ml Heparine, 25 U/ml Turbo DNase I, and Protease inhibitors). 400 μg of total RNAs were treated with RNAse I (250 U/l mg of total RNA) and nuclease digestion was stopped with SUPERaseIn RNase inhibitor. Monosomes were isolated using S-400 columns. Isolation of total RNA and mRNA protected fragments were 3 volumes of QIAzol® were added to the S-400 eluate before RNA purification with DIrect-Zol RNA Mini Prep Plus kit. RNAseq: The libraries were prepared with the following kit Ilumina stranded mRNA prep, ligation by the iGe3 genomics platform of the University of Geneva. Riboseq:  Selected RPFs (25-34 nt) were dephosphorylated with T4 Polynucleotide Kinase (NEB, #M0201S), the adapter Linker-1 was ligated to the 3' end of the RPF using T4 RNA Ligase 2 (NEB, #M0242S) and then purified. Ribosomal RNAs were substractes using RIboCop rRNA Depletion Kit V2 H/M/R. Reverse transcription was done by ProtoScript® II Reverse Transcriptase (NEB, #M0368S). Reverse transcriptase products were purifired and circulized with CircLigaseTM II ssDNA Ligase (Epicentre, #CL9021K). Final libraries were generated by PCR based on the forward index primer and one reverse index primers.