Single-nucleus RNA-seq of livers from mice fed standard or Western diet
Single-nucleus RNA-seq of livers from mice fed standard or Western diet
Animals were sacrificed by carbon dioxide inhalation at 38 weeks of age. To partially control for time-of-day effects, mice were sacrificed between 7:00 and 16:00 on two consecutive days, alternating between the four groups. Estrous cycle stage was not assessed in females. Mice were not fasted before organ collection. Half of each liver left lateral lobe was snap-frozen on liquid nitrogen, pulverised with a Covaris CP02 automated tissue pulverizer and stored at -80˚C as described previously (Panten et al. 2024). The whole liver was dissected out, and half of the left lateral lobe snap-frozen on liquid nitrogen. Mice were fed standard chow diet (Kliba # 3437) or WD (Research Diets # D16022301i; 40 kcal% non-transfat, 20 kcal% fructose and 2% cholesterol) for 32 weeks. Bcl2fastq was used to converts BCL files to paired-end .fq.gz files with PCR indexes in the header. Nuclei were released from cryopulverised liver tissue by homogenisation with a Qiagen TissueLyser III in hypotonic lysis buffer, resuspended in Sucrose PBS Triton X-100 MgCl2 (SBPSTM) buffer, fixed with MeOH/BS3, resuspended in SPBSTM and then stored at -80°C until use. Sci-RNA-seq3 library preparation was performed in 96 well plates by reverse transcription (first index), ligation (second index), second strand synthesis, protease digestion, tagmentation and PCR (third index), bead-cleanup and quality control.