Plasma samples were received on dry ice and stored at −80°C until processed. Prior to cfRNA extraction, plasma samples were thawed at room temperature and centrifuged at 1300 x g for 10 minutes at 4°C. cfRNA was extracted from plasma (300–1,000 μl) using the Norgen Plasma/Serum Circulating and Exosomal RNA Purification Mini Kit (Norgen, 51000). Extracted RNA was DNase treated with 14 μl of 10 μl DNase Turbo Buffer (Invitrogen, AM2238), 3 μl DNase Turbo (Invitrogen, AM2238), and 1 μl Baseline Zero DNase (Lucigen-Epicenter, DB0715K) for 30 minutes at 37°C, then concentrated into 12 μl using the Zymo RNA Clean and Concentrated Kit (Zymo, R1015).
Sequencing libraries were prepared from 8 μl of concentrated RNA using the Takara SMARTer® Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (Takara, 634485) and barcoded using the SMARTer® RNA Unique Dual Index Kit (Takara, 634451). Library concentration was quantified using the Qubit™ 3.0 Fluorometer (Invitrogen, Q33216) with the dsDNA HS Assay Kit (Invitrogen, Q32854). Libraries were quality-controlled using the Agilent Fragment Analyzer 5200 (Agilent, M5310AA) with the HS NGS Fragment Kit (Agilent, DNF-474–0500) and pooled to equal concentrations. Each pool was sequenced using both the Illumina NextSeq 500/550 platform (paired-end, 150 bp) and the Illumina NextSeq 2000 platform (paired-end, 100 bp).