RNA-seq of Homo sapiens: Fresh Tissue
For the isolation of nucleic acids from the tissue samples, commercial kits were used according to manufacturer instructions. Direct-zol RNA Miniprep was used for the extraction of fresh tissue (R2050, Zymo Research). Quick-DNA/RNA FFPE Miniprep was used for the extraction of FFPE samples (R1009, Zymo Research). Following RNA extraction, all downstream molecular biology steps were performed using our clinically validated metatranscriptomic method, as previously published (Hatch, A. et al. A Robust Metatranscriptomic Technology for Population-Scale Studies of Diet, Gut Microbiome, and Human Health. Int J Genomics 2019, 1718741 (2019).). The method includes DNase treatment, non-informative RNA depletion, cDNA synthesis, size selection, and limited cycles of PCR for adding dual unique barcodes to each sample. All samples were sequenced using the Illumina NovaSeq 6000 platform with 2x150 paired-end read chemistry.