Tumor-induced alterations in single-nucleus transcriptome of atrophying muscles

<h3><strong>Experimental Design</strong></h3> <p>Syngeneic C57BL/6 mice injected with Lewis Lung Carcinoma (LLC) cells were sacrificed 16 days after tumor inoculation while they experienced moderate cachexia and loss of muscle mass and function. We investigated single-nucleus transcriptomes of the tibialis anterior (TA) muscle from tumor-bearing mice and their non-tumor-bearing controls.</p> <h3><strong>Protocols</strong></h3> <p>Mice were housed at 22&deg;C and under 50% humidity with 12 hours of light and 12 hours of dark cycles (07:00 &ndash; 19:00) and given ad libitum access to a standard rodent chow diet and water in Koc University Animal Research Facility in accordance with institutional policies and animal care ethics guidelines.</p> <p>8-12 weeks old male mice with C57BL/6 background and Lewis lung carcinoma (LLC) cells were used for tumor inoculation. LLC cells were cultured in DMEM (Sigma, no. 5796) with 10% fetal bovine serum (FBS) and penicillin/streptomycin (Invitrogen). 5 million LLC cells were injected subcutaneously over the flank while control mice received PBS only. Muscle tissues were harvested at 16 days after LLC inoculation.</p> <p>TA muscle samples dissected from 6 mice in each group were combined and processed together. Samples were chopped with dissection scissors and placed into a lysis buffer containing 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, and 0.1% NP40 in nuclease-free water. Samples were homogenized using a douncer and filtered with 70 &micro;m and 40 &micro;m cell strainer. After centrifugation for 5 min at 500 g at 4 &deg;C, supernatant was discarded and nuclei were resuspended and stained with DAPI and subjected to fluorescence activated cell sorting.</p> <p>10X Genomics applications were used following the manufacturer&rsquo;s guidelines (Chromium Next GEM Single Cell 3ʹ Reagent Kits) to prepare the libraries, which were sequenced using the Illumina HiSeq X system.</p> <h3><strong>Data</strong></h3> <p>Sequencing data was first analyzed and filtered using Cell Ranger (v7.0) Single-Cell Software Suite provided by 10X Genomics. Data was counted and mapped with cellranger count function with --include-introns option for pre-mRNAs (mm10).</p> <p>Further analysis was performed using Seurat (v4.3.0) R (v4.2.2) package on R Studio (v2022.12.0), which filters nuclei, normalizes expression data, and carries out principal component analysis for clustering and Uniform Manifold Approximation and Projection (UMAP) visualization.</p> <h3><strong>Code</strong></h3> <p>You can see the code in R script file.</p>