transcriptome

Sample collection protocolHNSCC patients with primary oral (O) and oropharyngeal (OP) tumors treated at the Clinical Hospital Dubrava, Zagreb, Croatia between 2015 and 2017 were enrolled in the study. All patients underwent surgery as primary treatment and 2 pieces of tumor tissue were collected. Samples were stored in 700 μL of G2 lysis buffer (Qiagen) or RNA later (Ambion) solutions for the isolation of DNA and RNA, respectively. Samples were stored at +4 °C and delivered to the laboratory at the end of the day where they were stored at −20 °C for up to 2 weeks before DNA and RNA isolation.Nucleic acid extraction protocolFor nucleic acid extraction, smaller (~20 mg) pieces of tumor tissue were used. The DNA was isolated with the EZ1 Biorobot using EZ1 DNA tissue kit (Qiagen) following the manufacturer’s protocol. The total RNA was isolated using miRNeasy mini kit (Qiagen) also following the manufacturer’s protocol. Both DNA and RNA quality and quantity were analyzed on nanospectrofotometer (Implen). Furthermore, RNA quality was assessed by Bioanalyser RNA6000 Nano kit (Agilent) to determine the RIN number.Nucleic acid library construction protocolLibrary preparation was performed using TrueSeq Stranded mRNA (Illumina) kit.Nucleic acid sequencing protocolSequencing was performed on Nextseq500 instrument using NextSeq HIGH 150 cycles (Illumina) reagents on high output flow cell in paired end mode.high throughput sequence alignment protocolAlignment and quantification was performed with salmon v1.5.0 tool and the GRCh37 (2013 release) reference.