title: Illumina NovaSeq 6000 paired end sequencing; RNA-Seq of murine head and neck cancer cell line (MOC1) treated with antiPD-L1 antibody with or without an adenovirus encoding TNFa and IL-2
design: RNA-Seq of murine head and neck cancer cell line (MOC1) treated with antiPD-L1 antibody with or without an adenovirus encoding TNFa and IL-2
library_construction_protocol: Tumours were mechanically disrupted using gentleMACs C tubes (Milltenyi Biotech) followed by enzymatic digestion using collagenase type IV (1mg/mL), Hyaluronidase (2U/mL) and DNase I (10U/mL) in RPMI with 10% FBS for 1 hour. After double filtering of disrupted tumor tissue, CD45 Microbeads, mouse ( 130-052-301 Miltenyi Biotec) were used to isolate +/- fractions which were than placed into RNA later for isolation.  46 weeks old female C57BL/6JOlaHsd (immunocompetent) mice were purchased from Envigo (Indianapolis, IN, USA). Mice were subcutaneously (right flank) injected with either 2x106 MOC1 cells in 50l PBS. Established tumours (4mm)(stratified randomization) were treated with anti-PD-L1 Ab (clone 10F.9G2, BioXcell) (100 g/injection). When tumours reached 8mm (refractory), they were than treated with either intratumoral injection of PBS, 1  109 VP of unarmed control virus (Ad5-Luc), 1:1 mixture of Ad5-CMV-mTNF and Ad5-CMV-mIL2 virus (total 1  109 VP) or anti-PD-L1 Ab (clone 10F.9G2, BioXcell) (100 g/injection).  After extraction of gene hit counts, the gene hit counts table was used for downstream differential expression analysis. Using DESeq2, a comparison of gene expression between the customer-defined groups of samples was performed. The Wald test was used to generate p-values and log2 fold changes. Genes with an adjusted p-value < 0.05 and absolute log2 fold change > 1 were called as differentially expressed genes for each comparison. Below are the results of the number of significantly differentially expressed genes for all comparisons provided. RNA from CD45+/- MOC1 tumour cells was purified using RNeasy kit according to the manufacturers instructions (74106, Qiagen, Hilden, Germany) PolyA selection