Mutation analysis of HepaRG cells treated with Aflatoxin0.25, M 1
Differentiated No-Spin HepaRG cells (Lonza) were seeded at 4.8  10 cells/well in 24-well collagen-coated plates and cultured for 7 days in pre-induction medium to allow metabolic competence. Cells were maintained in Lonza Pre-Induction/Tox medium with appropriate supplements. Cells were exposed for 24 hours to test chemicals or vehicle (DMSO) in 2D monolayer culture. Post-exposure, media was replaced and cells were subjected to two sequential rounds of epidermal growth factor (EGF) stimulation over 7296 hours to promote fixation of mutations via cell division.Genomic DNA was isolated from exposed cells on Day 12 using the DNeasy Blood & Tissue Kit (Qiagen), following manufacturer instructions. DNA concentration and quality were assessed using a Qubit 4.0 Fluorometer and agarose gel electrophoresis.  Libraries were constructed using the TwinStrand DuplexSeq Mutagenesis Kit according to manufacturer protocols. Briefly, 500 ng of genomic DNA was ultrasonically sheared, ligated with unique molecular identifier (UMI) adapters, amplified using high-fidelity PCR, and subjected to hybrid capture targeting specific human genomic loci. Paired-end sequencing (2150 bp) was performed on an Illumina NovaSeq 6000 platform to a depth of >1,000 unique duplex coverage per sample.
SUB15539844
Aflatoxin0pt25_uM_HepaRG_1