2.5. RNA-Seq Library Preparation and Sequencing

Based on the preliminary qPCR results, besides CTRL and AFB1 experimental conditions, only cells exposed to the maximum concentration of R (i.e., 30 μM), either alone (R) or in combination with AFB1 (R+AFB1), were subjected to deep transcriptome investigations.

For each experimental condition, three independent biological replicates (i.e., independent cell culture experiments) were assessed. A total of 12 tagged RNA-seq libraries were prepared and sequenced following a 50 bp single-end strategy in an Illumina Hi-Seq 4000 instrument (Fasteris SA, Geneva, Switzerland). Libraries were prepared as previously reported [63]. Briefly, we used the Agilent’s SureSelect Strand Specific RNA Library Preparation Kit (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s instruction. Poly(A) mRNA was purified starting from 400 ng of total RNA and chemically fragmented. First-strand and second-strand cDNA were synthetized and end-repaired. Then, cDNA 3′ ends were adenylated and adaptors were ligated. Twelve cycles of PCR were used to amplify the adaptor-ligated cDNA library. The PCR products were then purified and size-selected using the SPRIselect reagent kit (Beckman Coulter, Brea, CA, USA). Library concentrations were measured adopting two approaches: Qubit RNA Assay kit (Life Technologies, Carlsbad, CA, USA), in a Qubit 2.0 Fluorometer (Life Technologies); PCR-based method using the NEBNext Library Quant Kit for Illumina (New England Biolabs, Ipswich, MA, USA). Individual libraries were monitored for insert size using the Agilent DNA 1000 assay kit (Agilent Technologies) on the Agilent Bioanalyzer 2100 instrument (Agilent Technologies).

Notably, libraries representing CTRL and AFB1 conditions have been previously analysed in a stand-alone study assessing the whole transcriptional effects of PCB126 and AFB1 on BFH12 cells [63]. In the present study, these libraries have been normalized and analysed again in the context of a larger dataset including new data (i.e., cells treated with R and R+AFB1).