GSM5574431: Liver Trit1 KO rep3 RPF; Mus musculus; RNA-Seq
50mg liver tissue powder was lysed in 1000 µl of ice-cold lysis buffer (10 mM Tris-Cl (pH 7.5), 300 mM KCl, 10 mM MgCl2, 1 mM DTT, 1% Triton X-100 and 10 U/ml DNAse I). Ribosome pellets were resuspended in Qiazol (Quiagen) after centrifugation of RNAseI digested monosomes (60 min at 25 °C and 1300 rpm on thermoblock) in 50% sucrose buffer, ribosome protected fragments were isolated using miRNAeasy kit (Quiagen) according to manufacturer protocol with modification to retain small RNAs. RPFs were electrophoresed on a 15% Tris-Borate-EDTA (TBE) Urea gel and RNA fragments between 15 and 40 nts in size were purified and incubated for 1 hout with T4 PNK according to manufacturers protocol. Small RNA sequencing libraries were constructed by GenXPro with the TrueQuant smallRNA kit with a depth of 30 M reads on an Illumina NextSeq500 machine. 50mg liver tissue powder was lysed in 1000 µl of ice-cold lysis buffer (10 mM Tris-Cl (pH 7.5), 300 mM KCl, 10 mM MgCl2, 1 mM DTT, 1% Triton X-100 and 10 U/ml DNAse I). Ribosome pellets were resuspended in Qiazol (Quiagen) after centrifugation of RNAseI digested monosomes (60 min at 25 °C and 1300 rpm on thermoblock) in 50% sucrose buffer, ribosome protected fragments were isolated using miRNAeasy kit (Quiagen) according to manufacturer protocol with modification to retain small RNAs. RPFs were electrophoresed on a 15% Tris-Borate-EDTA (TBE) Urea gel and RNA fragments between 15 and 40 nts in size were purified and incubated for 1 hout with T4 PNK according to manufacturers protocol. Small RNA sequencing libraries were constructed by GenXPro with the TrueQuant smallRNA kit with a depth of 30 M reads on an Illumina NextSeq500 machine.