AccuNext MNase Nucleosome DNA Library Preparation Kit

This product is a kit developed based on Micrococcal Nuclease (MNase), which is suitable for nucleosome localization and occupancy analysis, research on gene transcription and regulatory mechanisms, epigenetic and chromatin remodeling studies, exploration of disease mechanisms, and evolutionary and developmental biology applications. The starting volume of this product can be as low as 10⁴~10⁵Individual cells.
It utilizes the properties of micrococcal nuclease to selectively digest naked DNA and retain nucleosome-encapsulated DNA fragments for subsequent library construction and high-throughput sequencing analysis.
The reaction system of this product has been carefully optimized, all reagents should be used as provided in this product, it is not recommended to change any of the reaction components or use other equivalent products to replace the components in this product, in order to avoid obtaining bad experimental results. If substitution is required, please verify first.

 

1. High specificity: MNase Enzyme accurately digests naked DNA, preserving intact nucleosome DNA fragments (~150 bp sized monomer peaks are prominent).
2. Dual enzyme synergistic digestion: MNase Enzyme + Exonuclease III Enzyme is used in combination to reduce background noise and improve the resolution of nucleosome localization.
3. Optimization of the whole process: premixed buffer ensures the digestion efficiency, and DNA Clean Beads purification replaces phenol-chlorine extraction, which is safer to operate and has a higher purification recovery rate.

 

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

individual parts making up a compound	AG12572	AG12573
DNA Clean Beads	540 μl	1.7 ml

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

individual parts making up a compound	AG12572	AG12573
RNase A ( 10 mg / ml )	6 μl	20 μl
Proteinase K ( 20 mg / ml )	20 μl	60 μl
Lysis Buffer	200 μl	600 μl
Wash Buffer Ⅰ	1 ml x 2 pcs	1.5 ml x 4 pcs
1% Digitonin	4 μl	12 μl
Dilution Buffer	300 μl	1 ml
MNase Enzyme	4 μl	12 μl
Exonuclease III Enzyme ( 5 U/μl )	4 μl	12 μl
MNase Digestion Buffer	200 μl	600 μl
MNase Stop Buffer	24 μl	75 μl
10% SDS	24 μl	75 μl
Nuclease free water	1 ml	1 ml x 2 pcs

Note: Recommended use AccuNext DNA Library Preparation Kit (Illumina) (Code No. AG12535 / AG12536) is available for subsequent library construction, and can be purchased with the AccuNext CDI Junction Primers (Illumina, for DNA libraries) (Code No. AG12529 / AG12530 / AG12531),AccuNext Short junction primers (Illumina, for DNA libraries) (Code No. AG12537 / AG12538).

 

Storage temperature: Package 2-1 4℃ (avoid freezing)
Package 2-2 -20°C Storage
Transportation Temperature: Package 2-1 Ice Pack Transportation
Package 2-2 Dry Ice Transportation or -20℃ Ice Pack Transportation
Stability: Unopened reagents are valid for 12 months, after opening, they are stored according to the requirements of the components (e.g. MNase Enzyme is stored at -20℃ for ≤6 months after dispensing).

1. Use this product for 1×10⁴ K562 Cells were subjected to MNase fragmentation and the results of agarose gel electrophoresis were as follows:

2. Use the product for different cell volumes of K562 Cells (1×10).⁵5 x 10⁴1 x 10⁴) for library construction, the results showed that better sequencing data could be obtained for different cell volumes, with more concentrated fragment length distribution, normal TSS enrichment, better correlation of parallel samples as well as clear peaks and valleys in the IGV coverage map, as shown in the following figure:

a) Library fragment length distribution: fragment lengths are concentrated in ~150 bp size.

b) TSS region (near the transcription start site): shows an “inverted” or “depressed” morphology
Reason: nucleosome arrangement in promoter regions A very conserved pattern of nucleosome organization exists in the promoter regions of most classical genes.

• Nucleosome-Free Region (NFR): A region lacking nucleosomes usually exists near the transcription start site (TSS) of a gene. This open region provides binding sites for transcription machinery (e.g., RNA polymerase II) and various transcription factors, and is critical for initiating gene transcription.
• flanking nucleosomes: Upstream and downstream of the vacant region of the nucleosome, there is a regular arrangement of nucleosome-protected DNA segments. The first nucleosome immediately downstream of the NFR is often referred to as the +1 nucleosome, and the first one upstream as the -1 nucleosome.
  

c) Sample correlation analysis: 5 × 10⁴ Cells +100 U MNase group for example

d) IGV coverage map: positive control from https://ngdc.cncb.ac.cn/nucmap/samples.