Fastpure cell tissue dna isolation mini kit dc102

The 500 ng Cas12a plasmid, 250 ng crRNA plasmid, and 100 pmol of the double-stranded oligodeoxynucleotide (dsODN) GUIDE-seq tag were transfected into 24-well plate cells using 2.5 μl Hieff Trans Liposomal Transfection Reagent (CAT:40802ES03, Yeasen, Shanghai); 48 h after transfection, cells were harvested to extract genomic DNA using FastPure Cell/Tissue DNA Isolation Mini Kit DC102 (Vazyme, Nanjing). Approximately 1 μg of genomic DNA was sheared to a length of approximately 250 bp, added to a Y adapter, and amplified by 2 rounds of nested, anchored PCR. The last PCR products were GUIDE-seq libraries and sent to the company for sequencing using an Illumina sequencer.

Briefly, 600 ng Cas12a, 300 ng crRNA expression plasmids, and 10 pmol of the dsODN GUIDE-seq tag were transfected into a 24-well 293T cells (1.2 × 10⁵ cells per well); 48 h after transfection, the genomic DNA was harvested and purified using FastPure Cell/Tissue DNA Isolation Mini Kit DC102 (Vazyme). GUIDE-seq tag integration percentages and on-target modification were assessed by restriction-fragment length polymorphisms (RFLPs) assays and T7E1 assays (as described above), respectively. A total of 1,000 ng genomic DNA was fragmented, end repaired, A-tailing by using Hieff NGS Fast-Pace DNA Fragmentation Reagent (12609ES96, Yeasen, Shanghai). The sequencing library was prepared and sequenced on an Illumina Instrument and data was analyzed using guideseq v1.1 as described previously.