RNA-sequencing of Fresh-Frozen Head and Neck Tumor Samples

Fresh-frozen tumor samples were sectioned, collected for RNA preparation and in part subjected to tumor percentage evaluation by revision of HE stained coupes by senior head and neck pathologist Dr. S.M. Willems. Only samples with a tumor percentage of >40% proceeded to RNA-sequencing. RNA was isolated using the AllPrep DNA/RNA mini kit (Qiagen). Quality and quantity of total RNA was assessed by the 2100 Bioanalyzer using a Nano chip (Agilent, Santa Clara, CA). Only total RNA samples having RIN>7 were used for library preparation. Strand-specific libraries were generated using the TruSeq Stranded mRNA sample preparation kit (Illumina Inc., San Diego, RS-122-2101/2) according to the manufacturer's instructions (Illumina, Part # 15031047 Rev. E). The libraries were analyzed on a 2100 Bioanalyzer using a 7500 chip (Agilent, Santa Clara, CA), diluted and pooled equimolar into a 10 nM multiplex sequencing pool and sequenced with 65 base single reads on a HiSeq2500 using V4 chemistry (Illumina Inc., San Diego). Reads were mapped against the GRCh38 human genome using TopHat2.1 (70 (link)), with options “fr-firststrand,” “transcriptome-index,” and “prefilter multi-hits.” Read counts were determined using HTSeq-count (71 (link)) with options “stranded” and mode “union.”