Agilent 1290 infinity 2 liquid chromatography system

The prime editing guide RNA (pegRNA) was synthesized using Synthego’s CRISPRevolution platform with solid-phase phosphoramidite chemistry. Based on the original prime editing report [24 (link)], we selected a reverse transcriptase (RT) template of 10 nucleotides in length, inclusive of the C>G transversion, and a primer binding site (PBS) of 16 nucleotides in length. Three 2′-O-methyluridinylates were attached at the 3′ end of the PBS and stabilized with phosphorothioate backbones. The first three bases of the protospacer were modified as 2′-O-methyl derivatives and stabilized as phosphorothioates. The pegRNA was purified using reversed-phase high-performance liquid chromatography (Buffer A, 0.1 M TEAA; Buffer B, 50% 0.1 M TEAA/50% acetonitrile, 15%–95% B gradient in 15 min), and their identities were confirmed using an Agilent 1290 Infinity II liquid chromatography system coupled with Agilent 6530B Quadrupole time-of-flight mass spectrometry (Agilent Technologies, Santa Clara, CA) in a negative ion polarity mode.

For the serum samples, 50 µL samples were mixed with 300 µL mass spectrometry grade pre-chilled acetonitrile, then vortexed for 5 min. The mixture was then centrifuged at 15,000 × g and 4°C for 10 minutes, and the supernatant was collected. For fecal samples, 20 mg samples were weighed into a 2-mL screw top tube containing 50 mg of acid-washed glass beads, and then 120 µL mass spectrometry grade pre-chilled acetonitrile was added to each tube. The samples were homogenized under 70 Hz cryogenic grinding for 5 min. The tubes were then centrifuged at 15,000 × g and 4°C for 10 min, and the supernatant was collected. Measurements were obtained using an Agilent 1290 Infinity II Liquid Chromatography System coupled to an Agilent 6495A Triple Quadrupole Liquid Chromatography-Mass Spectrometry (LC-MS) System. Data analysis was conducted using MassHunter Workstation Data Acquisition, Agilent MassHunter VistaFlux Software, and Agilent Metabolite ID Software. The metabolites were identified based on the standards, MS/MS spectra, and the metabolite database METLIN (https://metlin.scripps.edu/indexphp).