Dntp mix

RNA was extracted from liver MTs and the cell suspensions obtained following the MT dissociation protocol at day 3 following standard TRIzol procedure with the addition of glycogen (LT-02241; ThermoFisher, Waltham, MA, USA). Extracted RNA was reverse transcribed using a M-MLV Reverse transcriptase (M1705; Promega,), M-MLV RT buffer (M531A; Promega), dNTP Mix (02-31-00100; Solis BioDyne), and Oligo dT-Primer (79237; Qiagen). The quantitative real-time polymerase chain reaction (qRT-PCR) was carried out using TaqMan probes (Table 2) for selected oxidative stress markers (HMOX1 and NQO1) and hepatic markers characteristic of HepaRG surrogates for hepatocytes (ALB), hTERT-HSC (CD44) and THP-1 surrogates for KCs (CD68). FastStart TaqMan Polymerase (04673433001; Roche) was used to perform the qRT-PCR. Program settings: 10 min denaturation at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The Ct values were generated using the Corbett Rotorgene Analysis Software 6000 and processed on GraphPad Prism. Gene expression changes were calculated using the ΔCt method with GAPDH as a house keeping gene. Fold changes were calculated as $2$ -(Δ(ΔCt) and expressed as mean and SD of 2 biological repeats with 3 replicates each.

Whole blood was collected to K2E (EDTA) vacutainers (Becton Dickinson) and donors’ genomic DNA was extracted from 1 ml of whole blood by the salting‐out method. The DNA sample purity and concentrations were measured by NanoDrop ND‐1000 spectrophotometry. Genomic DNA (500 ng) was treated with sodium bisulfite using the EZ DNA Methylation Kit (Zymo Research Corporation) according to the manufacturer's instructions. Bisulfite treated DNA was amplified in 10 ul reaction containing 0.72 ng of DNA, 1X Yellow PCR Buffer with (NH4)2SO4 (Naxo), 1.5mM MgCl₂ (Solis BioDyne), 2 mM dNTP mix (Solis BioDyne), 0.2 µM of each primer, and 0.06 U HOT FIREPol DNA Polymerase (Solis BioDyne). Cycle conditions were as follows: 95°C for 15 min, 1 cycle; 40 cycles (95°C for 20 s, 56°C for 30 s, 72°C for 1 min); and 72°C for 3 min, 1 cycle. Primer sequences used in PCR reactions can be delivered on request by the authors. Amplicons from each individual were combined in equal amounts, purified with Agencourt AMPure XP beads (Beckman Coulter) and labeled with Nextera XT v2 (Illumina) indexes. Paired‐end sequencing of bisulfite‐treated DNA with read length of 250 bp was done with Illumina MiSeq at the Core Facility of the Institute of Genomics of the University of Tartu.