title: RNA sequencing of human HNSCC cell lines
design: All samples were profiled by sequencing their exome DNA and their polyA+ mRNA transcriptome. Nucleic acids were extracted following lysis of cell pellets in 600 μl RTL lysis buffer (AllPrep DNA/RNA/miRNA Universal Kit, Qiagen). The lysates were homogenized in CK14 Precellyshomogenization tubes (Labgene Scientific) using the Minilys homogenizer (Bertin Technologies). DNA and RNA were purified from the homogenized lysates using a robotic workstation (Qiacube, Qiagen). following manufacturer’s instructions and their quality assessed using the Fragment Analyzer(Advances Analytical Technologies Inc., Ankeny, USA). RNA sequencing libraries were prepared using the Illumina TruSeq Stranded Total RNA reagents (Catalog number RS-122-2201; Illumina; San Diego,USA) according to the protocol supplied by the manufacturer and using 750 ng of total RNA. Exome sequencing libraries were produced as follows: 3 μg of genomic DNA was fragmented into 150-200 base pair fragments using the Covaris Sample Preparation System (S-series). Samples were prepared using SureSelect XT library prep kit (Agilent, Santa Clara, USA). The resulting adaptor-tagged libraries were then hybridized with exon biotin-coated baits from the SureSelect V5 kit (Agilent) for 24 hours at 65°C. Hybrids were captured with streptavidin-coated beads, PCR amplified and indexed.