FastDigest PteI

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	The purified DNA sample was treated in a 2-step reaction with a set of MREs in cocktail as RE1, including Cfr42I (SacII), PdiI (NaeI), Eco52I (EagI) and PteI (BssHII)(FastDigest enzyme from Thermal Scientific Inc.), called 4E collectively. Step 1 is the treatment with Cfr42I and PdiI in 1 × Tango Buffer at 37°C for 1 h. Step 2 is the treatment with Eco52I and Ptel after adjusting the buffer to 2 × Tango Buffer and incubating at 37° C for 1 h. Afterward, these MREs were heat-deactivated at 70°C for 10 min. The digested DNA sample obtained was immediately amplified using REPLI-g UltraFast Mini Kit (Qiagen) following the manufacturer-recommended protocol except that denature and neutralization steps were skipped.

Protocol tips

The purified DNA sample was treated in a 2-step reaction with a set of MREs in cocktail as RE1, including Cfr42I (SacII), PdiI (NaeI), Eco52I (EagI) and PteI (BssHII)(FastDigest enzyme from Thermal Scientific Inc.), called 4E collectively. Step 1 is the treatment with Cfr42I and PdiI in 1 × Tango Buffer at 37°C for 1 h. Step 2 is the treatment with Eco52I and Ptel after adjusting the buffer to 2 × Tango Buffer and incubating at 37° C for 1 h. Afterward, these MREs were heat-deactivated at 70°C for 10 min. The digested DNA sample obtained was immediately amplified using REPLI-g UltraFast Mini Kit (Qiagen) following the manufacturer-recommended protocol except that denature and neutralization steps were skipped.

Publication protocol

Cells were suspended and washed twice with phosphor-buffered saline (PBS). Single cells were picked up with a 1–10 ul pipette and dispensed into individual PCR tubes with each cell transferred with ∼1 μl volume of sheath PBS. Three microliters (3) genomic lysis buffer (Zymo Research) was then added to each tube and the cell was lysed in 10 min at room temperature. DNA in the lysate was purified by ethanol precipitation using Dr GenTLE precipitation carrier (ClonTech). The purified DNA sample was treated in a 2-step reaction with a set of MREs in cocktail as RE1, including Cfr42I (SacII), PdiI (NaeI), Eco52I (EagI) and PteI (BssHII)(FastDigest enzyme from Thermal Scientific Inc.), called 4E collectively. Step 1 is the treatment with Cfr42I and PdiI in 1 × Tango Buffer at 37°C for 1 h. Step 2 is the treatment with Eco52I and Ptel after adjusting the buffer to 2 × Tango Buffer and incubating at 37° C for 1 h. Afterward, these MREs were heat-deactivated at 70°C for 10 min. The digested DNA sample obtained was immediately amplified using REPLI-g UltraFast Mini Kit (Qiagen) following the manufacturer-recommended protocol except that denature and neutralization steps were skipped. The amplicons were combined with negative controls (no cells but PBS only), which did not show any visible band on electrophoresis gel. The amplified DNA was purified using genomic DNA clean & concentrator kit (Zymo Research). Usually approximately 3–4 μg of DNA amplicon was obtained, sufficient for PCR evaluation and library construction. This above was for the TEST sample. The control DNA (MC) was conducted through the same process except that no RE1 digestion steps were applied.

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