After nick-labeling, the samples were treated with Protease and IrysPrep Stop Solution (BioNano Genomics) [32 (link)]. The labeled DNA was stained with YOYO-1 (Invitrogen), and was loaded into the nanochannels following the established protocol [33 (link)]. We generally collected 60x coverage (180Gb) data to obtain 30 molecules containing the telomeres for each chromosome. The image analysis was done following the established procedure [32 (link)].
Irysprep stop solution
Manufactured by Bionano Genomics
Sourced in United States
The IrysPrep Stop Solution is a laboratory reagent used in sample preparation for genomic analysis using Bionano Genomics' technology. Its core function is to halt the DNA labeling process during the IrysPrep workflow.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using irysprep stop solution
1
Telomere Visualization by Optical Mapping
2
Whole Genome DNA Imaging and Analysis
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The DNA samples were treated with Protease and IrysPrep Stop Solution (BioNano Genomics, San Diego, CA, USA) [20 (link)]. The labeled DNA was stained with YOYO-1 (Invitrogen, Carlsbad, CA, USA) before being loaded into the nanochannels following a previously established procedure [24 (link)]. A total of 180 Gb data, which is about 60× coverage, provided around 30 molecules with telomeres for each chromosome arm. The image analysis was performed following the established method [20 (link)].
3
High-throughput Genomic Mapping Pipeline
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After nick-labeling with either the two- or three-color schemes, the samples were treated with 6 mAU of QIAGEN Protease at 56°C for 30 min, and the reaction was stopped with 1 µL of IrysPrep stop solution (BioNano Genomics). The DNA backbone was stained with 333 nM YOYO-1 (Invitrogen) and is shown in blue in all figures. The stained samples were loaded and imaged inside the nanochannels following the established protocol (Lam et al. 2012 (link)). BioNano Genomics labeling kit and IrysChip were used to generate the nick labeling data. The next generation mapping system from BioNano has dramatically improved the throughput; our custom-made systems are very similar to this new BioNano Genomics system. Each IrysChip contains two nanochannel devices, which can generate at least 60 Gb of data (molecules >150 kb). Normally, 60× coverage (180 Gb) is needed to generate 30 molecules of each chromosome end containing the telomeres. This assay runs 3 d, collecting over 24,000 images. It currently costs $1000 per sample to run whole-genome mapping. The image analysis was done using BioNano commercial software for segmenting and detecting DNA backbone based on the YOYO-1 staining similar to early optical mapping method (Lin et al. 1999 (link)), and localizing the green labels by fitting the point-spread functions.
4
DNA Nicking using Cas9 and Nt.BspQI
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For DNA nicking using the 48 and 162 sgRNA mix (supplementary Tables S1 and S2 ),1.25 μM of the synthesized sgRNA was first incubated with 125 nM of Cas9 D10A (NEB) in 1× NEBuffer 3.1 (NEB) at 37°C for 15 min to form a sgRNA-Cas9 complex. 300 ng of the DNA sample was then added to the sgRNA–Cas9 complex mixture and incubated at 37°C for 60 min. For DNA nicking with both Cas9 and Nt.BspQI, 2.5 μM gRNA was first incubated with 63 nM of Cas9 D10A in 1X NEBuffer 3.1 at 37°C for 15min. After that, 300 ng of DNA and 5 U of Nt.BspQI (NEB) were added to the sample mixture and incubated at 37°C for 2 h. The nicked DNA samples were then labeled using 5 U Taq DNA Polymerase (NEB), 1× thermopol buffer (NEB), 266 nM free nucleotides mix (dATP, dCTP, dGTP (NEB) and Atto-532-dUTP (Jena Bioscience)) at 72°C for 60 min. The labeled sample was then treated with Proteinase K at 56°C for 30min and 1uM IrysPrep stop solution (BioNano Genomics) was added to the reaction.
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