Posttranscriptional reprogramming controls MASLD progression through chronic ER stress adaptation (RNAseq 2)
Posttranscriptional reprogramming controls MASLD progression through chronic ER stress adaptation (RNAseq 2)
A piece of approximately 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 minutes. Supernatants were collected and RNA was extracted with Trizol, the resulting mRNAwas loaded in a ploy(U) chromatography. 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. mRNA fractionation was performed using poly(U) chromatography, as described in Belloc and Mendez 2008. RNA sequencing was conducted on the input sample and on fractions 4 and 5, which correspond to the 55°C and 65°C elutions, respectively. mRNA were then extracted with Trizol 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.