title: cell-free RNA (cfRNA) sequencing of Homo sapiens: plasma
design: Total cfRNA was isolated from 200 L of plasma utilising the miRNeasy Serum/Plasma Advanced Kit (Qiagen), which employs Qiagens unique resin as the separation matrix. This kit is capable of extracting all RNA molecules from approximately 18 nt upwards. DNase I (NEB) was used to eliminate remaining DNA.  To assess potential contamination in the RNA extraction and library preparation processes, two types of negative controls were utilised. These included libraries generated from RNA/DNA-free water (Invitrogen) and solutions eluted from the miRNeasy Serum/Plasma Advanced Kit with RNA/DNA-free water as input. A non-reverse transcriptase control was implemented to prevent DNA contamination. The positive control comprised human brain RNA, provided SMARTer Stranded Total RNA-Seq Kit, and was processed using the same protocol as that for the cfRNA library preparation.  The comprehensive cfRNA library (approximately > 50 nt) was constructed using the SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian (Takara), adhering to the manufacturer's recommended protocol. This kit employs a CRISPR/DASH technique to eliminate ribosomal cDNA. Each library was sequenced using Illumina NovaSeq6000 platform using a PE150 strategy.