Xenograft Transcriptome Analysis Pipeline

RNA was extracted from white and thermogenic implants using the TRIzol method. Library preparation was performed using the TruSeq cDNA library construction (Illumina). Samples were processed on the Illumina HiSeq 550 sequencing system with the NextSeq 500/550 High Output kit v2.5 (Illumina, Cat. No. 20024906). The generated fastq files were loaded into the DolphinNext platform (https://dolphinnext.umassmed.edu/) and the Bulk RNA sequencing pipeline was used. The. fastq files were aligned to both the human (hg38) and the mouse (mm10) genome. The resulting alignments were processed using the R-package XenofilteR to classify reads as either of human or mouse origin. Reference (https://github.com/PeeperLab/XenofilteR; Netherlands Cancer Institute - Genomics Core Facilty, 2022 ; Kluin et al., 2018 (link)) for more details on XenofilteR source code. Once aligned, the files were run through RSEM for normalization. Differential expression analysis was performed using the DEBrowser platform (https://debrowser.umassmed.edu/). Gene ontology analysis was performed by combining results from TopFunn (https://toppgene.cchmc.org/).