Quantifying Cellular Nucleotide Levels

dNTP and rNTP concentration (pmol/million cells) for activated CD4⁺T cells were collected from a previous study having 4 replicates, and the precalculated average was used to represent dNTP/rNTP ratios (41 (link)). The nucleotide samples were extracted based on the established protocol (42 (link)) with some modifications. To prepare the nucleotide samples, 2 × 10⁶ cells of HEK293T or hESC-h9 were counted and centrifugated to obtain a cell pellet. The pellet was washed with PBS and then vortexing was performed for 2 min with 200 μl of cold 65% methanol for cell lysis. The cell mixture was incubated at 95°C for 3 min and then incubated on ice for 1 min to complete the cell lysis. By centrifugation at 14 000 rpm for 3 min, the supernatant containing nucleotides was isolated. To quantify the intracellular dNTPs and rNTPs, an ion pair chromatography-tandem mass spectrometry method (43 (link)) was applied, with modifications. Chromatographic separation and detection were performed on a Vanquish Flex system (Thermo Fisher Scientific) coupled with a TSQ Quantiva triple quadrupole mass spectrometer (Thermo Fisher Scientific). Analytes were separated using a Kinetex EVO-C18 column (100 × 2.1 mm, 2.6 μm) (Phenomenex) at a flow rate of 250 μl/min. Pmol/million cells were calculated for rNTPs and dNTPs for all four replicates to calculate the ratios.