title: GSM8891456: Plasma cell-free RNA, RNA seq for subject: cfs_cfrna_163, ME/CFS patient; Homo sapiens; RNA-Seq
library_construction_protocol: Plasma was stored at -8-˚C and then thawed at room temperature. Then ~500 μL samples were aliquoted and centrifuged at 3000 x g for 8 minutes at 4˚C. cfRNA was extracted using the Norgen Plasma/Serum Circulating and Exosomal RNA Purification Mini Kit (Norgen, 51000).  Extracted RNA was DNase treated with a combination 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 the Illumina NovaSeq 6000 platform (paired-end, 150 bp).