Fig. 2

a Optimization of DNA extraction from rare and fixed cells using Qiagen QIAamp DNA Micro Kit. ~200–300 cells/experiment, N ≥ 2 per condition. ① Fresh cells using Cell Protocol yielded 6–7 pg of DNA/cell and were used as a control. ② Cell Protocol did not work on cells fixed with 4% PFA. ③ Tissue Protocol significantly increased the DNA yield when Proteinase K digestion was extended from 4 to 36 h, with significant advantage of overnight incubation. ④ Increasing the Proteinase K digestion temperature from 56 to 60 °C further increased the DNA yield from 60 to 80%. ⑤ The optimized protocol was then verified on fixed, fixed+permeabilized and fixed+permeabilized+stained HCT116 cells to mimic the output from CTC enrichment platforms. b Validation of an optimal DNA extraction protocol for low number of fixed cells. Different amounts of HCT116 cells fixed with 4% PFA were seeded inside a 96 well-plate, imaged and counted. DNA was extracted from these cells using the optimized protocol defined in A (Qiagen QIAamp DNA Micro Kit, Tissue Protocol, Proteinase K digestion overnight at 60 °C) and quantitated using qPCR