To assess the NHEJ activity, two systems were adopted. For Cas9-mediated cleavage, the Cas9/sgHPRT-expressing vector and double-stranded DNA oligonucleotides Ins (25 pmol, Thermo Fisher Scientific) (34 (link)) together with siRNAs, GapmeRs or SMG6-expressing vectors were transfected into HeLa cells. Genomic DNAs were collected 48 h post-transfection and extracted by the PureLink™ Genomic DNA Mini Kit (Thermo Fisher Scientific). Total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific) and then subjected to reverse transcription with SuperScript III (Thermo Fisher Scientific). For qPCR, the reactions containing 100 ng of genomic DNA or cDNAs, specific primers and PerfeCTa SYBR Green FastMix PCR Reagent (Quanta Biosciences) were performed in a LightCycler 480 Real-Time PCR System (Roche). For a GFP-based reporter assay, siRNAs, GapmeRs or SMG6-expressing vectors and the pSCE expression plasmid were transfected into HeLa GFP reporter cells (TopoGEN). Cells were harvested 72 h post-transfection. GFP-positive cells were detected by fluorescence-activated cell sorting using 1a 7-color LSR II Analytic Flow Cytometer (BD Biosciences).
Perfecta sybr green fastmix pcr reagent
Manufactured by Quanta Biosciences
The PerfeCTa SYBR Green FastMix PCR Reagent is a pre-mixed, ready-to-use solution for quantitative real-time PCR (qPCR) amplification of DNA sequences. It contains SYBR Green I dye, hot-start DNA polymerase, buffers, and stabilizers optimized for fast, highly sensitive, and specific real-time PCR.
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2 protocols using perfecta sybr green fastmix pcr reagent
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Quantifying NHEJ Activity in Cells
2
NHEJ Activity Assay in HeLa Cells
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HeLa cells were maintained as above for U2OS cells. To assess the NHEJ activity, 1 µg of the Cas9/sgHPRT expressing vector (sgHPRT targeting sequences: AAAGGGTGTTTATTCCTCA), 25 pmol of dsDNA oligonucleotides Insertion (Ins dsDNA-1: 5′-TTAATTGAGTTGTCATATGTTAATAACCGG-3′; Ins dsDNA-2: 5′-ACCGTTATTAACATATGACAACTCAATTA-3′, Du et al. 2018 (link)), 40 pmol Y14 targeting siRNA (5′-ggguauacucuaguugaaa-3′, Thermo Fisher Scientific) and 4 µg of siRNA-resistant Y14-expressing vectors were transfected into 2 × 105 HeLa cells (Chuang et al. 2023 (link)). Genomic DNA was collected 48 h post-transfection and extracted by PureLink Genomic DNA Mini Kit (Thermo Fisher Scientific). For qPCR, the reactions containing 100 ng genomic DNA, specific primers, and PerfeCTa SYBR Green FastMix PCR Reagent (Quanta Biosciences) were performed in a LightCycler 480 Real-Time PCR System (Roche). The primers used were Cas9/sgHPRT forward (5′-ATCCAATCAAATGTTTGTATCCTGT-3′), Ins (5′-GAGTTGTCATATGTTAATAACGG-3′) and reverse (5′-CCCTTCAATGTTTACTTTGTTCTGG-3′).
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