Author:
Becker Caroline,Aygar Sema,Daheron Laurence
Abstract
AbstractCopy number increases involving chromosome 20q with gain of the geneBCL2L1are a prevalent form of genomic instability in hPSC. In addition to large aneuploidies, findings in this region often include microamplifications that are too small to detect by G-banded karyotyping. Gene editing procedures warrant especially close monitoring of 20q genomic stability because they involve p53-activating stressors that select for the survival ofBCL2L1-aneuploid cells. Here we describe an optimized strategy for detectingBCL2L1copy number increases in hPSC cultures using duplexed droplet digital PCR (ddPCR) with genomic DNA or cell lysate as the starting material. The procedure consists of droplet generation, thermocycling, droplet reading and data analysis. The expected result is a copy number estimate derived by comparing the number of droplets positive forBCL2L1to the number positive for a reference template,PVRL2. This procedure generates same-day screening results for 1 to 96 samples, providing a convenient option for screening hPSC cultures that is easily integrated into a gene editing workflow.
Publisher
Cold Spring Harbor Laboratory