Abi hidi formamide

Sanger sequencing method was performed to screen mutations in the UBA1 gene considered the pathognomonic alteration of VEXAS syndrome and not included in our t-NGS panel. Fifty ng of DNA sample were used for the first amplification reaction (Polymerase Chain Reaction, PCR) of the specific target region of the UBA1 gene (exon 2), using primers reported in Supplementary Table 2. PCR productions were purified using GeneUP ExoSAP kit (Applied Biosystems, Waltham, USA) and 2 µl of purified products were used for subsequent Sequencing PCR following the manufacture’s protocol of BigDye^® terminator v3.1 cycle sequencing kit (Applied Biosystems, Waltham, USA). Sequencing reaction products were purified using the CentriSep columns (Applied Biosystems, Waltham, USA) according to the manufacturer's recommendations. Samples were diluted with 16 µl of ABI HiDi Formamide (Applied Biosystems, Waltham, USA) and resolved on ABI 3130 automated sequencer (Applied Biosystems, Waltham, USA). Sequence Scanner Software 2.0 was used to analyze the results.