title: Illumina HiSeq 2000 sequencing; RNAseq of the two isogenic variants of the CAL27 head-and-neck cancer cell line with wild-type or mutated CYP1B1 genotype
design: RNAseq of the two isogenic variants of the CAL27 head-and-neck cancer cell line with wild-type or mutated CYP1B1 genotype
library_construction_protocol: The  HNSCC cell line with no detectable CYP1B1 expression, CAL27, was infected. 2×105 cells were infected with viruses bearing the pER51CYP1B1-WT vector, or the pER51CYP1B1-VAR vector, or the pER15 control vector (p15), at a multiplicity of infection (MOI) of 10 or 40. Cells were then washed and amplified in 25 cm2 flasks and selected by puromycin (2 µg/ml) for further analysis.  RNA was extracted using the RNeasy blood and Tissue kit (Qiagen) according to the manufacturer's instructions. The quantity and quality of each RNA sample were measured spectrophotometrically using Nanodrop 1000 (Thermo Scientific, Waltham). The wild-type human CYP1B1 cDNA was purchased from Open Biosystems and was cloned in a pDONR201vector using the Gateway Cloning kit (Invitrogen). An attB-flanked PCR product of CYP1B1 cDNA was cloned into the pDONR201 entry vector and then transferred into destination vector pSD69 using the Gateway Cloning kit (Invitrogen). The pSD-69 plasmid contains the human phosphoglycerate kinase (PGK) promoter, a Gateway attR cassette (Invitrogen), the mouse PGK promoter and the puromycin acetyltransferase gene cloned into pRRLhPGK.GFP.SIN18.14 The variant cDNA was obtained by site-directed mutagenesis through replacement of G1697 by C1697, using the Site-Directed Mutagenesis kit (Stratagene). Constructions were verified by sequencing and nucleotide replacement was checked by pyrosequencing. The pER51CYP1B1-WT (G allele: WT) and pER51CYP1B1-VAR (C allele: VAR) plasmid DNAs were then used for the generation of viruses and the infection of cell lines. The tdTomato-CAL27 and -CAL33 isogenic cell lines were produced by transduction of the cells with a second lentivirus carrying the tdTomato reporter gene (PGK-tdTomato) which was a generous gift from Pr François Moreau-Gaudry (University of Bordeaux).