title: Illumina NovaSeq 6000 paired end sequencing; RNA-seq of livers from female mice treated for eight weeks with the glucagon analogue, NNC9204-0043, compared to female mice treated with PBS (control group)
design: RNA-seq of livers from female mice treated for eight weeks with the glucagon analogue, NNC9204-0043, compared to female mice treated with PBS (control group)
library_name: Sample 22_p
library_construction_protocol: The mice were fasted overnight (11 hours) prior to liver isolation, and livers were kept at -80°C until analysis. For eight weeks, female C57BL/6JRj mice (seven weeks old at the start of treatment) received, by subcutaneous injections, either a long-acting glucagon analogue (GCGA, NNC9024-0043, 1.5 nmol/kg body weight, terminal half-life: 5-6 hours) or PBS + 1% BSA (PBS). Dose and administration frequency of GCGA was determined in a pilot study, in which 3 nmol/kg or 30 nmol/kg GCGA or PBS was administered to male and female C57BL/6JRj mice (8 weeks old) once or twice during a 24 hour period. The dose of GCGA was initially 3 nmol/kg body weight, but after 16 days of treatment, one of the mice had lost >20% of its body weight and had to be terminated (this mouse was excluded from the study). Due to this effect of GCGA, the dose was halved (1.5 nmol/kg body weight) for the remainder of the study. Raw sequencing data were processed using bcl2fastq software version 2.20.0 (Illumina). Total DNA/RNA was purified by an AllPrep DNA/RNA Minikit (80204, QIAgen) according to manufacturer's instructions and quality tested using a 2100 Bioanalyzer instrument (Agilent Genomics). The extract was DNase treated with RNeasy Minikit (74106, QIAgen) and RNase-Free DNase set (79254, QIAgen) according to manufacturer's protocol. All the above-mentioned processes were performed on a QIACube machine (QIAgen).  Library preparation was done using TruSeq Stranded Total RNA Library Prep Gold (20020599, Illumina) and IDT for Illumina-TruSeq RNA UD Indexes (20022371, Illumina) according to the manufacturer's instructions. Some samples (input <100 ng) were prepared with NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB-E7760S, New England Biolabs), NEBNext rRNA Depletion Kit 24 with beads (NEB-E7765, New England Biolabs), and NEBNext Multiplex Oligos for Illumina (E7600S, New England Biolabs).