GSM9588323: Hepa1-6, Eef1d exon 5 KO clone #3, RNA-seq, replicate 2; Mus musculus; RNA-Seq
For Ribo-seq, ~3 million cells were lysed in 400 μl of a lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 1 mM dithiothreitol, cycloheximide (100 μg/ml), chloramphenicol (100 μg/ml)), and the lysate was treated with 15 U of Turbo DNase (Thermo Fisher Scientific) and then cleared by centrifugation. For RNA-seq, RNA was extracted from the same lysate used for Ribo-seq using Trizol LS (Thermo Fisher Scientific) and Direct-zol RNA Microprep Kit (Zymo Research), then treated with the NEBNext rRNA Depletion Kit v2 (NEB) to deplete ribosomal RNAs. The Ribo-seq library was prepared according to a protocol described previously (Methods 126, 112-129, 2017 and STAR Protoc 1, 100168., 2020). The RNA-seq library was prepared using a NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB).