CRISPR knock-in validation

Cultured hippocampal neurons tagged with oScarlet were prepared with Lucigen

QuickExtract DNA extraction solution (Biosearch Technologies, cat# QE09050).

Briefly, neurons were lysed by adding 50 ul of QuickExtract solution to each

sample.

The samples were mixed by pipetting and incubated at 68 °C for 15 min

followed by 95 °C for 10 min in a thermocycler before being stored at -20 °C

for downstream analysis.

Two different PCR reactions were performed to amplify

DNA products of ~500 bp corresponding to the 5’ and 3’ integration junctions.

PCR#1 used the α-syn forward primer: TGTGCTTTCTCTTCCCTCTCTG and the reverse oScarlet primer CCGTCCTCGAAGTTCATCAC, whereas PCR#2 used the α-syn forward primer:

ATAACACTTCGTGCAGCACC and the reverse oScarlet primer ACAGGATGTCCCAGGAGAAG.

PCR products were extracted from the agarose gel using the Monarch DNA gel

extraction kit (New England Biolabs, cat# T1020S), and samples were submitted for

sequencing for analysis at MCLAB.