GSM3580989: WT_RNA_seq_1hRecovery-4; Mus musculus; RNA-Seq
Cells were rapidly washed twice with ice-cold PBS containing 50 μg/ml of cycloheximide. 5% of the clarified lysate was used for RNA-seq. 30 μg of RNA were used to isolate ribosome-protected fragments following RNase I digestion. After RNase digestion, lysates were loaded on a sucrose gradient and centrifuged at 100,000 rpm to recover ribosome-protected fragments. RNA from the resuspended ribosomal pellet was purified and run on a gel to selectively excise foot-printed RNAs (from 17 nt to 34 nt RNA fragments). To avoid any ribosomal contamination in the library preparation steps, we then performed Ribo-Zero Gold depletion of the foot-printed RNA. The rRNA depleted RNA fragments were dephosphorylated and the linker was added. To specifically deplete unligated linker, yeast 5'-deadenylase and RecJ exonuclease digestion was performed. Subsequent library preparation steps were performed essentially as previously in Linder B., et. al. Nat Methods 12, 767-772 (2015). To avoid any ribosomal contamination in the library preparation steps, we then performed Ribo-Zero Gold depletion of the foot-printed RNA. The rRNA depleted RNA fragments were dephosphorylated and the linker was added. To specifically deplete unligated linker, yeast 5'-deadenylase and RecJ exonuclease digestion was performed. Subsequent library preparation steps were performed essentially as previously in Linder B., et. al. Nat Methods 12, 767-772 (2015). Library preparation was performed using the NEBNext Ultra Directional RNA Library Prep Kit starting from 1 ug of total input RNA and following the protocol for use with NEBNext rRNA Depletion Kit.