Posttranscriptional reprogramming controls MASLD progression through chronic ER stress adaptation (RNAseq)
Posttranscriptional reprogramming controls MASLD progression through chronic ER stress adaptation (RNAseq)
A piece of aproximately 30 mm3 of snap frozen murine liver was homogenized in lysis buffer [20 mM Tris-HCl pH 7.8, 10 mM MgCl2, 100 mM KCl, 1% Triton X-100, 2 mM DTT, 100 μg/ml CHX, complete protease inhibitors] using a bead beating grinder.  Lysates were passed through a 26G needle (BD bioscience) for further homogenization, and centrifuged at 4°C 1300xg for 10 min. Supernatants were collected and used to perform Ribosome profiling (RP) and total mRNA isolation, as previously described in Slobodin et al. 2017. Male C57BL/6J mice were housed under standard conditions. At 6 weeks of age, mice were randomly assigned to either a high-fat diet (HFD) or a normal diet (ND) and fed HFD or ND for 30 weeks when the experiments were perfomed. RNA was isolated using TRI-sure reagent (Bioline) according to the manual, using Glycoblue (Ambion) as a carrier. RNA libraries were prepared for sequencing using the kit NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (Cat#E7770), following manufacturer´s instructions and 10-11 cycles of amplification.