RNAseq from cuticles of tumor free (w1118) and tumor bearing Drosophila larvae (MARCM RasV12, scrib1) tumour-bearing Drosophila larvae

To investigate which factors might be driving the cachectic muscle wasting observed in our larval models bearing RasV12, scrib1 tumors, we performed RNA sequencing (RNAseq) on the cuticles of wild type and RasV12, scrib1 tumour-bearing larvae. Pathway and gene ontology analysis revealed significant deregulation of stress and starvation response genes, metabolic genes, and inflammation and immune regulatory genes within cachectic muscle of tumour bearing larvae. Cuticles were dissected in freshly autoclaved PBS and all internal tissues were removed to keep only the body wall including the skeletal muscles (40 cuticles/sample). Cuticles were frozen in a collection tube placed on dry ice. Total RNA was extracted using QIAGEN RNAeasy kit, following the manufacturer’s instructions, including the on-column DNase digestion step. For RNA-seq an RNA integrity score was determined to check RNA quality C11(Agilent technologies 2200 Tapestation, RNA Screen Tape).