Mutation Analysis of KLF2 and NOTCH2

Nested PCR was performed using Platinum SuperFi DNA polymerase (Thermo Fisher Scientific). To amplify exon 1–2 and exon 3 of KLF2 and exon 34 of NOTCH2, the PCR conditions were 10 s at 98 °C (30 s for the first cycle), followed by 20 cycles of 30 s at 58 °C and 1.5 min at 72 °C (5 min for the last cycle) for the first round of PCR and 30 cycles for the second round of PCR. Amplification products were electrophoresed on 2% agarose gel and stained with ethidium bromide. Presence of somatic mutations in the KLF2 (exon 1–3) and in the NOTCH2 (exon 34) were investigated by Sanger sequencing as described^{7 (link), 9 (link), 10 (link)}. The primer sequences used for nested PCR and sequencing analysis are listed in Supplementary Table S2.