GSM5860394: Day0_2_RNAseq; Mus musculus; RNA-Seq
For each sample, around 5-10 million cells were used for both Ribo-seq and RNA-seq. The cells were first washed with cold ice-cold PBS supplemented with 100 μg/ml cycloheximide and then were lysed for 5 minutes on ice in polysome lysis buffer (5mM MgCl2, 150mM NaCl, 20mM Tris-Cl (pH=7.5), 1% Triton X-100, 1 mM dithiothreitol, 20 U/ml DNase I, 100 μg/ml cycloheximide and nuclease-free H2O). Using immediate repeated pipetting we dissociated cells to create a homogeneous lysate. Lysates were next centrifuge at 10,000g at 4°C for 5 minutes to precipitate cell debris and the supernatant was quantitated by Qubit. To accurately dissect translational control, both Ribo-seq and RNA-seq libraries were prepared for each biological replicate from the identical lysate by Ribomaps Ltd. One aliquot of the lysate (10%) was supplemented with RNseq inhibitor and processed for RNA-seq library generation. Remaining lysate (90%) was treated with RNase. Then ribosome protected mRNA fragments were isolated and used to generate Ribo-seq library.