Affi gel boronate gel

Measurement of nucleotide pools was performed as described in (47 (link)). Briefly, cells at OD₆₀₀ ∼0.4 were rapidly (<3 min) harvested by filtration (MF-Millipore Membrane Filter, mixed cellulose esters, 0.8 μm, Sigma-Aldrich/Merck AAWP02500), for a total yield of ∼7.4 × 10⁸ cells. rNTPs and dNTPs were extracted in an ice-cold mixture of 12% (w/v) TCA and 15 mM MgCl₂, and neutralized with an ice-cold freon–trioctylamine mixture (10 ml of freon [1,1,2-trichloro-1,2,2-trifluoroethane], Millipore Sweden AB [>99%], and 2.8 ml of trioctylamine, Sigma-Aldrich Sweden AB [98%]). 575 μl of the aqueous phase were pH adjusted with 1 M ammonium carbonate (pH 8.9), loaded on boronate columns (Affi-Gel Boronate Gel, Bio-Rad), and eluted with 50 mM ammonium carbonate (pH 8.9)- 15 mM MgCl₂ mix to separate dNTPs from rNTPs. The purified dNTP eluates were adjusted to pH 3.4 and analyzed by HPLC on a LaChrom Elite^® HPLC system (Hitachi) with a Partisphere SAX HPLC column (Hichrome, UK). rNTPs were directly analyzed by HPLC in a similar way as dNTPs by using 24 μl aliquots of the aqueous phase, adjusted to pH 3.4.

Acetonitrile was obtained from Merck (Darmstadt, Germany), while N-glycosidase F (PNGase F, 500 U/μL) was obtained from New England Biolabs (Ipswich, USA). Trypsin was sourced from Beijing Shengxia Proteins Scientific Ltd. (Beijing, China). The bicinchoninic acid protein assay kit and HeLa protein digest standard were acquired from Pierce (Rockford, USA). Unless specified otherwise, all other chemical reagents were obtained from Sigma-Aldrich (St Louis, USA). Distilled water was purified using a Milli-Q system (Millipore, Milford, USA). Ultrafiltration tubes (MWCO=10 kDa) were purchased from Millipore (Amicon Ultra; Merck), and MonoSpin C18 was obtained from Shimadzu (Kyoto, Japan). Affi-Gel boronate gel was sourced from Bio-Rad Laboratories (Hercules, USA).