title: GSM5484315: Liver_HF_rep1_Ribo; Mus musculus; OTHER
library_construction_protocol: RNA-seq: Total RNA samples were prepared from two independent experiments (HF and HB groups). Total RNA was isolated using TRIzol® RNA extraction reagent (Ambion, Austin, Texas, USA), following the manufacturer's instructions. Equal amounts of total RNA from each preparation were pooled for subsequent library construction and RNA-seq. Ribo-seq: A total of 90 mg of liver tissue was pre-treated with 100 mg/ml cycloheximide for 15 min, followed by three washes with pre-chilled PBS prior to the addition of 2 ml of cell lysis buffer [1% Triton X-100 in ribosome buffer (RB buffer, 20 mM HEPES-KOH (pH 7.4), 15 mM MgCl2, 200 mM KCl, 100 μg/ml cycloheximide, and 2mM dithiothreitol)] to T-75 flasks. After 30-min in an ice-bath, cell lysates were split into two pre-chilled 1.5 ml tubes. Cell debris was removed by centrifugation at 16,200 × g for 10 min at 4 °C. Supernatants were transferred into new pre-chilled 1.5 ml tubes with 2 μl of Ribolock RNase Inhibitor (40 U/μl, Fermentas) in each tube. RNase I (10 U/μl, Fermentas) was then added at 0.2 μl per tube, followed by incubation for 15 min at 37 °C, and the reaction terminated with 1% SDS (1/10 volume per tube). The digested samples were pooled and layered onto the surface of 15 ml of sucrose buffer (30% sucrose in RB buffer). The ribosomes were pelleted by ultracentrifugation at 185,000 × g for 5 h at 4 °C. RNA extraction was then performed by the Trizol method and ribosomal RNA (rRNA) depleted with a Ribo-Zero rRNA Removal Kit (Human/Mouse/Rat) (Epicenter) by the manufacturer's instructions. The fraction with an insertion size ~28 nt was excised and purified from a gel. RNA-seq: The sequencing libraries were constructed following the TruSeqTM RNA Sample Preparation Guide (Illumina, San Diego, California, USA).   Briefly, the polyA+ mRNA was selected with RNA Purification Beads (Illumina).   The library was constructed by using the Illumina TruSeq RNA sample Prep Kit v2 and sequenced with an Illumina HiSeq-2000 for 50 cycles.   High quality reads that passed the Illumina quality filters were kept for sequence analysis.  Ribo-seq:RPFs were sequenced with an Illumina HiSeq-2000 sequencer for 36 or 50 cycles.