title: GSM2823264: RNA-seq for MYC(Tg); KRAS(G12D) mouse liver tumor 2; Mus musculus; RNA-Seq
library_construction_protocol: Mouse liver tumours were micro-dissected immediately after animal euthanization. Liver perfusion (with ice cold PBS containing 10 mg/ml cycloheximide (CHX)) and placed in ice cold PBS containing 10 mg/ml CHX for 5 min. Tumours were minced into small pieces with a scalpel and incubated for 20 min in 0.5% collagenase supplemented with 10 mg/ml CHX. After spun down, tumour was resuspended in 4 ml trypsin containing 5 mg/ml CHX for 10 to 15 min at 37 °C, followed by filtering through a 40 m cell strainer and resuspension in the lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 15 mM MgCl2, 100 μg/ml cycloheximide, 1 mM DTT, 0.5% Triton X-100, 0.1 mg/ml heparin, 8% glycerol, 20 U/ml TURBO™ DNase, 1x Combined Protease and Phosphatase Inhibitor) All Ribo-Seq libraries were prepared as described in Ingolia, N. T., Brar, G. A., Rouskin, S., McGeachy, A. M. & Weissman, J. S. After removing the nuclei and mitochondria, lysate was treated with RNase A/T1 mix (0.5 mg RNase A (Ambion, AM2272) and 1,000 U RNase T1 (Life Technologies, 2280)) to digest mRNAs that are not protected by the ribosome. The digestion was stopped by adding SUPERase-In™ RNase Inhibitor (20 U/μl, Ambion, AM2696), followed by centrifuging to pellet ribosomes. Ribosome-protected fragments were purified by TRIzol®, and two biological replicates were performed for each type of Ribo-Seq and all Ribo-Seq libraries (three biological replicates for WT liver).
attributes.tag: GEO Accession
attributes.value: GSM2823264