Amicon ultra 0.5 30k

Directly after the PCR, Uracil-DNA glycosylase (NEB, USA) (2.5 U) was added to the PCR solution and the entire mixture was then gently mixed via pipetting. After incubation at room temperature for 5 min the mixture was heated to 95°C for 5 min and then cooled back to room temperature; this resulted in the formation of a 9 nucleotide long toehold sequence. The PCR-UDG mixtures were purified using an Amicon Ultra-0.5 30K (Millipore, USA) according to the recommendations of the manufacturer. The nucleic acids were then eluted with a displacement 1× TEM buffer (pH 8), consisting of Tris·HCl (10 mM), EDTA (1 mM) and MgCl₂ (12.5 mM). Quantification of the eluted PCR products was carried out using a NanoDrop 1000 spectrophotometer (Thermo Scientific, USA).

For protein processing, a shotgun bottom-up approach was applied. In summary, a 30 µL volume of each sample was prepared after the normalization in the previous step to obtain a final concentration of 1.5 µg/µL per sample. An equal volume of reduction solution (DTT 0.1 M) was added to each sample and then incubated for 40 min at 56°C. Using the FASP method [47 (link)], the samples were processed. This technique utilizes a filter with a nominal molecular weight limit of 30,000 (Amicon Ultra-0.5 30 k, Millipore). After transferring the samples into the FASP filters, alkylation step was done using IAA solution (0.05 M) for 20 min in the dark at room temperature. Digestion was then carried overnight at an incubation temperature of 37°C using LysC/trypsin (40 µg/mL in 50 mM Tris-HCL solution at pH 8). The filters containing the digests were then rinsed using 50 µL of saline solution (0.5 M) and the enzyme activity was stopped with 10 µL of TFA 5% for each tube. Enrichment and desalting were then performed for each sample with a ZipTip C-18 (Millipore) before undergoing LC-MS/MS analysis.