Targeted RNA-Seq of homo sapiens: adult plasma cell-free RNA
Cell-free nucleic acids were extracted from plasma using the QIAamp Circulating Nucleic Acid kit and subjected to DNase I digestion (RNase-Free DNase Set, Qiagen) to isolate cell-free RNA. Cell-free RNA libraries were generated using double-stranded cDNA synthesis (NEBNext Ultra II) followed by end repair, adapter ligation, and amplification (KAPA Hyper Prep). Targeted capture with a custom gene panel targeting rare abundance genes (RAGs) was performed using the Twist Biosciences Hybridization kit. Libraries were sequenced using paired-end 150 bp reads on Illumina HiSeq4000, NovaSeq6000, and NovaSeqX instruments.