GSM7508089: monosome Chow, rep 2; Mus musculus; OTHER
Liver pieces from control diet-fed or High-Fat-High-Sugar (HFHS) WT mice were collected and flash-frozen in liquid nitrogen. Using 200mg of frozen livers, lysates and ribosome footprints were generated by RNase I digestion and purified according to Janich etal. (2015) with minor modifications. Briefly, 10 μg of RNA was separated and excised from a 15% urea-polyacrylamide gel (30nt or 60nt footprints -monosomes or disomes respectively). From the gel slices, RNA was extracted overnight at 4 °C on a rotating wheel before precipitation in isopropanol during 3h at -20 °C RNA3'-end repair was carried out with 2 U/μl of T4 PNK (Lucigen) prior to a 2h adaptor ligation at 25°C using 1.5 μl of ligase mix (0.33 U/μL of T4 RNA Ligase 1 (NEB), 6.66 U/μl of T4 RNA Ligase 2 Deletion Mutant (Lucigen) and 1.33 U/μL of riboguard (Lucigen)) and 1 uL of a 20 μM 5'-adenylated DNA adaptor (containing sample barcode and a 8N unique molecular identifiers (UMIs)). Adaptor removal was carried out by treating individual libraries with 2.7 U/μl of 5'deadenylase (NEB) and 1.8U/μL of RecJf exonuclease (NEB) for 1h at 30°C and 1h at 37°C. Using Zymo Clean & Concentrator columns, samples were purified and pooled . Ribosomal RNA was depleted according to siTools Biotech rRNA depletion kit specifications with a custom-made riboPOOL. The clean-up step was done using Zymo Clean & Concentrator columns