The DNA was labeled with nick-labeling [47 (link)] as described previously using the IrysPrep Reagent Kit (BioNano Genomics). Specifically, 300 ng of purified genomic DNA was nicked with 7 U nicking endonuclease Nt.BspQI (New England BioLabs, NEB) at 37°C for two hours in NEB Buffer 3.1. The nicked DNA was labeled with a fluorescent-dUTP nucleotide analog using Taq polymerase (NEB) for one hour at 72°C. After labeling, the nicks were ligated with Taq ligase (NEB) in the presence of dNTPs. The backbone of fluorescently labeled DNA was stained with YOYO-1 (Invitrogen).
A subset of the samples were labeled using the newer Direct Label Enzyme (DLE) method (Bionano Genomics). These samples used the DNA Labeling Kit-DLS 80005 and followed the manufacturer’s instructions. In Summary, 750ng of the gDNA was labeled using DLE-1 enzyme and reaction mix followed by Proteinase K digestion (Qiagen). The DNA back bone was stained after drop dialysis. The stained sample was then homogenized and incubated at room temperature over nigh before quantified using Qubit dsDNA HS Kit (Invitrogen).
A subset of the samples were labeled using the newer Direct Label Enzyme (DLE) method (Bionano Genomics). These samples used the DNA Labeling Kit-DLS 80005 and followed the manufacturer’s instructions. In Summary, 750ng of the gDNA was labeled using DLE-1 enzyme and reaction mix followed by Proteinase K digestion (Qiagen). The DNA back bone was stained after drop dialysis. The stained sample was then homogenized and incubated at room temperature over nigh before quantified using Qubit dsDNA HS Kit (Invitrogen).
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