Serum RNA Extraction and Sequencing

RNA was extracted from 200 µl of sera using the Agencourt RNAdvance blood extraction kit (Beckman Coulter) following manufacturer’s instructions and involving a DNase treatment step at 37°C for 15 minutes as previously described.¹ (link) Extracted RNA was converted to cDNA using Superscript III (Invitrogen) and random hexamers. The cDNA was converted to double-stranded DNA using the NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs). Library preparation was performed using the KAPA LTP Library Preparation Kit from Kapa Biosystems, and the process was automated using the Biomek FXP liquid handler from Beckman Coulter. NEBNext Multiplex Oligos were utilized for indexing and amplification.¹¹ The amplified libraries were quantified using a Qubit 3.0 fluorimeter from Invitrogen and a 4200 TapeStation from Agilent. The libraries were then pooled in equal molar amounts and sequenced using the NextSeq 500/550 High Output v2.5 300 cycle kit, employing a paired-end read configuration with a length of 2 × 150 bp. In order to lower the chance of cross-contamination, a stringent one-way sequencing system with separated rooms for each stages of the NGS sequencing process was followed.