RNA extraction from PBMCs was performed following the instructions of the RNeasy RNA blood mini kit (QIAGEN). RNA from PAXgene blood tubes was extracted using the PAXgene Blood RNA System (Qiagen) following the manufacturer’s protocol. RNA abundance was measured photometrically (Xpose), and RNA integrity number (RIN) was determined (Agilent Bioanalyzer).
Cancer-related transcripts were enriched and sequenced according to the method described in detail in ref 25 . Briefly, 50 ng total RNA were reversely transcribed with oligo-dT using kit SQK-PCB109 from Oxford Nanopore Technologies and amplified by PCR. cDNAs were captured with a custom Agilent SureSelect probe set directed against 123 hereditary cancer genes. Following a second PCR amplification of captured cDNAs, sequencing libraries were prepared with the SQK-PCB109 kit and sequenced on R9 flow cells on the GridION. Capture and sequencing was performed in triplicates. Differential expression analysis has been performed against 21 reference samples in triplicates using the Oxford Nanopore Technologies Pipeline for differential gene expression (DGE) and differential transcript usage analysis of long reads (https://github.com/nanoporetech/pipeline-transcriptome-de ), which uses minimap2 V.2.18,21 (link) salmon V.1.5.026 (link) and edgeR V.3.34.0.27 (link)
Cancer-related transcripts were enriched and sequenced according to the method described in detail in ref 25 . Briefly, 50 ng total RNA were reversely transcribed with oligo-dT using kit SQK-PCB109 from Oxford Nanopore Technologies and amplified by PCR. cDNAs were captured with a custom Agilent SureSelect probe set directed against 123 hereditary cancer genes. Following a second PCR amplification of captured cDNAs, sequencing libraries were prepared with the SQK-PCB109 kit and sequenced on R9 flow cells on the GridION. Capture and sequencing was performed in triplicates. Differential expression analysis has been performed against 21 reference samples in triplicates using the Oxford Nanopore Technologies Pipeline for differential gene expression (DGE) and differential transcript usage analysis of long reads (
