AccuNext CUT & RUN DNA Library Building Kit (Illumina)

This product is developed for Illumina high-throughput sequencing platform, based on the principle of CUT & RUN (Cleavage Under Targets and Release Using Nuclease) technology to study the interaction between protein and DNA. With optimized reaction system and library construction process, this product has the advantages of high experimental success rate, antibody compatibility, short experimental period, simple operation and high signal-to-noise ratio compared with traditional ChIP-Seq, which is especially suitable for epigenetic research fields such as early embryonic development, stem cells, tumorigenesis and so on.
CUT & RUN is a new method to study protein-DNA interaction, using Protein A/G fusion MNase nuclease to precisely target to the target protein under the guidance of antibody and fragmentation of DNA near the target site, the extracted DNA can be directly used for the construction of DNA libraries, and the DNA information of the target site can be obtained after sequencing.
The reaction system of this product has been carefully optimized, all reagents in the experiment should be used as provided in this product, it is not recommended to change any reaction components or replace the components in this product with other equivalent products in order to avoid obtaining bad experimental results. If replacement is needed, please verify it first.

 

1. For illumina sequencing platform: integrates Index and illumina connectors and constructs DNA libraries that can be directly sequenced by NGS.
2. High compatibility: from 1 X 10³ ~ 5 X 10⁵ A starting volume of cells was experimented with.
3. High signal-to-noise ratio: low background noise signal, good experimental repeatability.
4. Simple operation: the whole process of magnetic bead extraction and purification, simple and rapid operation.
5. Strong performance of pAG-Mnase: pAG structural domain has broad-spectrum affinity for antibodies from rabbit, mouse and other sources, with precise targeting; optimized Mnase enzyme can efficiently and accurately cleave the target DNA.
6. High quality sequencing: optimized PCR amplification system, high quality sequencing data.

 

Package 2-1 is organized as follows (stored at 4°C):

individual parts making up a compound	AG12562	AG12561
ConA Beads Pro	20 μl	60 μl
DNA Clean Beads	470 μl	1.5 ml
10% SDS	8 μl	24 μl

Package 2-2 is composed as follows (stored at -20°C):

individual parts making up a compound	AG12562	AG12561
Lysis Buffer	400 μl	1.2 ml
10X ConA Binding Buffer	100 μl	300 μl
10X Wash Buffer	700 μl	1 ml X 2 pcs
5% Digitonin Solution	8 μl	24 μl
10X Dig-300 Buffer	220 μl	660 μl
BE Buffer	14 μl	42 μl
pAG-Mnase	4 μl	12 μl
CaCl2 Solution	8 μl	24 μl
Stop Buffer	6 μl	18 μl
Proteinase K (20 mg / ml)	4 μl	12 μl

Storage temperature: Package 2-1 4°C (avoid freezing)
Package 2-2 -20℃ Storage
Transportation Temperature: Package 2-1 Ice Pack Transportation
Package 2-2 Dry Ice or -20°C Ice Pack Transportation

1. The amount of cells used for library construction of K562 Cells with this product is 10⁴The number of amplification cycles was 13, and the library was detected by Agilent 2100 Bioanalyzer using H3K27me3 antibody, resulting in a typical Ladder-like distribution of peaks, as shown in the figure below:

Figure 1: Agilent 2100 Bioanalyzer library length detection

2、Using this product for different amount of cells (1 X 10) ⁴1 X 10 ⁵5 X 10 ⁵ Cells) K562 Cells were used for library construction with H3K27me3 antibody, and the results showed that good sequencing data could be obtained for different cell volumes, with TSS enrichment as well as a high signal-to-noise ratio in the IGV view, and significant enrichment for the target gene loci, as shown below:

Figure 2: TSS enrichment map

Figure 3: chr2 IGV global view