22 Life Technologies
™
| Parkinson's cell models
After analyzing the TaqMan
®
SNP Genotyping Assay results from each plating of cells, Ion PGM
™
sequencing was then performed
on candidate colonies to assess clonality and to characterize the second allele deletion. For the final selected clonal population,
sequencing showed that the original 2 bp deletion was now present in all cells, and half of the sequences lacked an additional
6 bases flanking these 2 bases (Figure 10). The identified deletions were further verified by Sanger sequencing.
Figure 10. Ion PGM
™
sequencing of the edited iPSC clone with SNCA
-/-
. (A) Percent of reads containing a deletion shown as a function of nucleotide position. (B) IGV software clearly
indicates an 8 bp deletion on the second allele.
Nucleotide Percent deleted
1 51
2 51
3 99
4 99
5 52
6 40
7 39
8 39
A B
Generation of α-synuclein knockout lines from the MSA line (continued)
1 2 3 4 5 6 7 8
SNCA
-/ -
edit – IGV viewer
SNCA
-/-
edit as shown in IGV
8 7 6 5 4 3 2 1
The QuantStudio
®
12K Flex Real-Time PCR System is an all-in-one instrument designed for
maximum throughput, flexibility, and scalability. The QuantStudio
®
12K Flex system was used to
determine the presence of specific mutations in each colony (Figures 4 and 9) and to assess the
pluripotency status of double mutant colonies (Figure 11C).
Learn more at lifetechnologies.com/quantstudio12k
Instrument highlight—QuantStudio
®
12K Flex Real-Time PCR System
