Thermal block

PCR-RFLP genotyping for the two loci was performed as follows. The target region of the NUDT15 and TPMT genes was amplified by PCR using the primers PCP-0023/24 and PCP-0027/28 with the stepdown PCR protocol as described above. Each NUDT15 c.415C>T genotyping restriction digestion reaction contained 1.7 µL unpurified PCR product, 0.2 µL FastDigest Taal restriction enzyme (Thermo Fisher, Waltham, MA, USA), 0.3 µL 10× FastDigest Green Buffer (Thermo Fisher, Waltham, MA, USA) and nuclease-free water added up to 5 µL. Each TPMT*3C genotyping restriction digestion reaction contained 1.7 µL unpurified PCR product, 0.2 µL AccI restriction enzyme (New England Biolabs, Ipswich, MA, USA), 0.3 µL CutSmart Buffer (New England Biolabs, Ipswich, MA, USA) and nuclease-free water added up to 5 µL. The digestion mix was incubated either at 65 °C (for TaaI digestion) or 37 °C (for AccI digestion) for 15 min on a thermal block (Eppendorf, Hamburg, Germany), and separated alongside 2.0 µL of undigested PCR product using a 2% agarose gel in 1× TBE buffer by electrophoresis at 100 V for 45 min.

RNA was isolated using TRIzol reagent. After five minutes at room temperature, chloroform was added, and the vials were shaken and incubated for three minutes at room temperature. The vials were then centrifuged (Allegra 64R Centrifuge, Beckman Coulter, Brea, CA, USA) at 4 °C for 15 min at 12,000× g. The upper phase was extracted from the vial, and isopropanol was added. After 10 min at room temperature, the samples were centrifuged again at 4 °C at 12,000× g for 10 min. The supernatant was discarded, and the RNA pellet was washed twice with 75% ethanol diluted with diethyl pyrocarbonate (DEPC) water. Then, the pellet was centrifuged again at 7500× g and 4 °C for five minutes. The ethanol supernatant was discarded, and the pellet was resuspended in DEPC water. The samples were dissolved at 58 °C for 10 min in a thermal block (Eppendorf, Hamburg, Germany). RNA concentration, A 260/230 and A 280/260 values were detected using the Nanoquant plate (Tecan, Männedorf, Switzerland) and the Tecan GENios microplate reader (Infinite M Plex, Tecan). Samples were stored at −80 °C until cDNA synthesis.

Random 10 nt long DNA sequences with balanced base distributions were generated using the bgen tool (gear.embl.de). Barcoded PCR primers were functionalized with a 5′-end biotin for purification, followed by a spacer sequence, a common primer sequence, a unique barcode sequence, and a ligation site (Table 2). The reverse complements (RC) were functionalized with a free 5′-end phosphate to enable ligation. Barcoded RT primers comprised a dT(20)-VN sequence, a unique barcode sequence, and a phosphate group at the 5′-end. The RC for this had a ligation site complementary to the ligation site of the PCR primers. A list of all barcode sequences can be found in the supplementary materials. Complementary sequences were annealed at equimolar concentrations by heating mixtures to 95 °C for 10 min in a thermal block (Eppendorf) followed by their cooling to room temperature (RT) for 1 h.Table 2

Sequences used in the barcode fragments

BC-PCR	5′-biotin-TTTTTTTAAGCAGTGGTATCAACGCAGAGTACNNNNNNNNNNgcggc RC: 5′-Phos-NNNNNNNNNNGTACTCTGCGTTGATACCACTGCTTAAAAAAA
BC-RT	5′-[Phos]NNNNNNNNNNTTTTTTTTTTTTTTTTTTTTVN RC: 5′-NNNNNNNNNNgccgc