C9 (Group 4)
The RNA-Seq of each individual sample was carried out from IGA Technology Services (Udine, Italy). cDNA libraries were constructed with 400 ng of total RNA by using the Illumina NovaSeq 6000 Universal Plus Total RNA-Seq with NuQuant kit (Tecan Trading AG, Mnnedorf, Switzerland) following the manufactures instructions. The workflow consists of fragmentation of total RNA and cDNA synthesis with a mixture of random and oligo (dT) primer, followed by end repair to generate blunt end, ligation of UDI adaptors, strand selection, AnyDeplete to remove unwanted transcript, such as ribosomal RNA, and PCR amplification to generate the final library. Total RNA samples and final cDNA libraries were quantified with the Qubit 2.0 Fluorometer (Invitrogen, Carlsbadm CA, USA) and quality tested by Agilent 2100 Bioanalyzer Nano Assay. Sequencing was carried out in paired-end mode (150 bp) by using NovaSeq 600 (Illumina) with a targeted sequencing depth of about 80 million reads per sample.