Total RNA was extracted from FFPE tissue sections using a single phenol/chloroform extraction protocol with Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNase-free DNase set (Qiagen, Courtabouef, France) was used to remove DNA. The DNase was eliminated by a second Trizol extraction. RNA was quantified by NanoDrop (Thermo Fisher Scientific, Cheshire, UK) and quality was controlled (DV200 value cutoff > 13%) by TapeStation with Hs RNA Screen Tape (Agilent, Courtaboeuf, France).
For each sample, 100 ng of total RNA was used to prepare libraries with TruSeq RNA Access Library Prep Kit (Illumina, San Diego, USA). 14 libraries were pooled at a DNA concentration of 4 nM with 1% PhiX. Sequencing was performed (75 cycles paired end) with NextSeq 500/550 High Output V2 kit on a NextSeq 500 instrument (Illumina). Data analysis was performed on BaseSpace Sequence Hub (Illumina) with the RNA-Seq Alignment application. Alignments to the human reference genome (hg19) were performed with STAR26 (link) and TopHat227 (link). Fusion transcripts were identified with Manta28 (link) and TopHat2 fusion.
For each sample, 100 ng of total RNA was used to prepare libraries with TruSeq RNA Access Library Prep Kit (Illumina, San Diego, USA). 14 libraries were pooled at a DNA concentration of 4 nM with 1% PhiX. Sequencing was performed (75 cycles paired end) with NextSeq 500/550 High Output V2 kit on a NextSeq 500 instrument (Illumina). Data analysis was performed on BaseSpace Sequence Hub (Illumina) with the RNA-Seq Alignment application. Alignments to the human reference genome (hg19) were performed with STAR26 (link) and TopHat227 (link). Fusion transcripts were identified with Manta28 (link) and TopHat2 fusion.
