Bulk RNA-sequencing of normal and Kaposi sarcoma tissues from skin and gastrointestinal tract

Matched tissue samples from 19 patients were harvested for total RNA and analyzed by bulk RNA-sequencing. Tissues were stored in RNAlater and lysed with Trizol. Tissues were homogenized and extracted for total RNA with Direct-Zol Miniprep kit (Zymo). Ribosomal RNA was removed, and sequencing libraries were prepared using NEBnext Ultra Low Input Total RNA with rRNA Depletion Prep (NEB). The reads from the FASTQ files (reads are generated by HiSeq Illumina 4000 and NovaSeq 6000) were trimmed of Illumina adapters usingcutadapt with default parameter except for --q 10 and--minimum-length 25. We generated a ‘combined genome reference’ by catenating the human reference genome (GRCh38) and KSHV reference genome (NC009333). The reads (average 45.9 million reads per sample) were aligned to the combined genome reference using STAR (version 2.7.8a). The transcriptome bam files created by STAR was used to estimate the read counts and Transcript per million (TPM) values at the gene level using RSEM v1.3.2. The GENCODE v30 reference gene annotation (GTF) combined with the KSHV gene annotation were used for annotation using STAR and RSEM.