Optimized enzymatic cell lysis protocols for single-cell genomic analysis

Area of Science:

• Molecular Biology
• Genomics

Background:

• Optimizing cell lysis is critical for accurate single-cell genomic analysis.
• Current enzymatic lysis protocols often require long incubation times, delaying downstream applications.

Purpose of the Study:

• To reduce enzymatic lysis incubation time for single-cell genomic analysis.
• To maintain genomic DNA integrity and amplification efficiency under optimized conditions.

Main Methods:

• Human leukocytes were subjected to proteinase K digestion.
• Incubation durations ranged from 2-16 hours at temperatures between 37-60 °C.
• Genomic DNA recovery was assessed using Y-chromosome-specific PCR.

Main Results:

• Shorter incubation periods (e.g., 2-4 hours) at elevated temperatures (60 °C) yielded comparable or superior PCR amplification efficiencies to overnight digestion at 37 °C.
• No significant differences in DNA yield or quality were observed between optimized and standard lysis conditions.
• Statistical analysis confirmed the robustness of the optimized protocol.

Conclusions:

• Enzymatic lysis duration can be significantly reduced without compromising DNA integrity or yield.
• The optimized protocol facilitates same-day genomic preamplification, increasing single-cell workflow throughput.
• This approach offers practical benefits for clinical diagnostics and forensic genetics.