Cetuximab and Nivolumab in Recurrent/Metastatic Head and Neck Cancer
Total RNA was assessed for quality using the Caliper LabChip GX2. The percentage of fragments with a size greater than 200nt (DV200) was calculated using software. An aliquot of 850 ng of RNA was used as the input for first-strand cDNA synthesis using Illumina's TruSeq RNA Exome kit. Synthesis of the second strand of cDNA was followed by indexed adapter ligation. Subsequent PCR amplification enriched for adapted fragments. The amplified libraries were quantified using an automated PicoGreen assay . 200ng of each cDNA library, not including controls, were combined into 4-plex pools. Capture probes that target the exome and IDT's xGen® Blocking Oligos were added to reduce off target binding. The samples and reagents were hybridized for the recommended time. Following hybridization, streptavidin magnetic beads were used to capture the library-bound probes from the previous step. Two wash steps effectively remove any nonspecifically bound products. These same hybridization, capture and wash steps are repeated to assure high specificity. A second round of amplification enriches the captured libraries. After enrichment the libraries were quantified with qPCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms and then pooled equimolarly. The entire process is in 96-well format and all pipetting is done by either Agilent Bravo or Hamilton Starlet