title: GSM7984487: Arabidopsis, +ORF1, +ORF2-Cas9, replicate 3; Arabidopsis thaliana; OTHER
library_construction_protocol: High molecular weight DNA was isolated from fresh Arabidopsis seedlings and Soybean leaf tissue using the NucleoBond HMW DNA kit (Takara), and digested by restriction enzymes XbaⅠ and AluⅠ. Fragmented DNAs ranging from ~450bp to ~2kb were purified from agarose gels, A-tailed by Klenow Fragment (3'-5' exo-; NEB), and ligated to the pGEM T- Easy vector (Promega). 1 µL of this ligation product was used as a template for primary PCR, followed by secondary PCR using nested primers. mPing specific primers were used with the pGEM T- Easy vector primers for primary and secondary PCRs. Barcoded sequencing adapters were added to the amplicons through their inclusion in the PCR primers. The sequencing library for soybean was constructed similarly, except that the genomic DNA was isolated from leaf tissue and digested by XbaⅠ, PmlⅠ, and AluⅠ. After passing quality controls, libraries were run on a MiSeq (Illumina) with V2 output (1X300)