Illumina NovaSeq 6000 paired end sequencing; RNA-seq of gastrocnemius muscle of orthotopic lung cancer mice with cachexia and sham control.
Immune competent, 11 weeks old male 129S2/Sv mice, were randomly allocated to either (1) sham control group or (2) tumor-bearing (OLCC) group. Syngeneic lung epithelium-derived adenocarcinoma cells (K-rasG12D; p53R172HΔG) were inoculated intrapulmonary into the left lung lobe of the mice. Body weight and food intake were measured daily. At baseline and weekly after surgery, grip strength was measured and tumor growth and muscle volume were assessed using micro cone beam CT imaging. After reaching predefined surrogate survival endpoint, animals were euthanized and skeletal muscles of the lower hind limbs were collected forRNA sequencing.Gastrocnemius muscle was collected and snap frozen in liquid nitrogen. Muscle was grinded into powder and homogenized in TRI Reagent by using a Mini bead beater (Cole Parmer) sample homogenizer, and total RNA was extracted. TRI Reagent (Sigma-Aldrich) was used according to the manufacturers' protocol NEXTFLEX Poly (A) Beads 2.0 kit (PerkinElmer)