title: NextSeq 500 paired end sequencing; LINE-1 methylation as survival predictive factor in patients with stage III-IV oropharyngeal squamous cell carcinoma
design: LINE-1 methylation as survival predictive factor in patients with stage III-IV oropharyngeal squamous cell carcinoma
library_construction_protocol: We retrospectively reviewed a cohort of 156 patients with stageIII IVB (AJCC TNM 7 th Ed.) OPSCC. Forty patients were diagnosed and treated at Treviso Regional Hospital, and 116 at National Cancer Institute of Aviano and/or Pordenone Hospital. All patients were managed with curative intent with elective radiotherapy ± chemotherapy or surgery ±adjuvant (chemo)radiotherapy, in accordance with current clinical practice. Genomic DNA was extracted from OPSCC formalin-fixed paraffin-embedded tissues. The quantification of HPV16DNA was performed by real-time quantitative PCR by using specific primers for the amplification of a region spanning the E6 genes of the HPV16 genome, whereas the methylation status of LINE1repetitive sequences was evaluated by real-time quantitative Methylation-Specific PCR. RNA extraction and purification from treated cells were performed using SNET lysis buffer (10 mM Tris pH 8.0, 0.1M EDTA, 0.5% sodium dodecyl sulphate) and phenol/chloroform/isoamyl Nucleic acids were quantified with Qubit 2.0 fluorimeter using Qubit RNA HS assay kit (Termo Fisher Scientifc, USA) and the assessment of nucleic acids integrity (RNA Integrity Number) was performed with Agilent 4150 TapeStation System (Agilent Technologies, USA).  Libraries preparation for transcriptome analysis was performed employing the TruSeq RNA Exome kit (Cat.20020189, Illumina) for FFPE samples starting from 200 of RNA as input materials, respectively, according to manufacturersprotocols