A new method for targeted genomic insertions of large DNA sequences is demonstrated in human pluripotent stem cells for constructs up to 173 kb NBTHighlight
variants. To improve target specificity, 2–5 locked nucleic acids were included per probe. Amplification conditions for each probe were optimised for discriminating amplicons containing that specific missense mutation. Droplets were analyzed using the “absolute quantification” option of the QuantaSoft software.transcripts from the wildtype allele and the allele reintroduced by STRAIGHT-IN, probes were developed to detect a heterozygous synonymous mutation present in exon 6.
For each cell line, 500 ng of gDNA was fragmented by sonication, followed by Illumina library prep using the KAPA Hyper Kit and unique barcoding of each sample with IDT adapters . The samples were purified twice to remove any adapter dimers and fragments below 150bp. The adapter ligated samples were further PCR enriched using the p5 and p7 primers for 10x cycles using the KAPA HiFi mastermix.
The Hyb Module box of the SureSelectQXT Target Enrichment for the Illumina Platform was used for hybridization and capturing of each sample separately. Briefly, 750 ng of the Illumina-prepped sample and 500 ng of the biotinylated RNA capture probes were used in the reaction. A 15 cycle post capture PCR with the p5 and p7 primers was performed using the KAPA HiFi mastermix, with the resulting samples purified using 0.8x Ampure XP beads and quantified using the Qubit 2.
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