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Nucleosomes consisting of approximately 146 base pairs (bp) of DNA wrapped around a histone octamer are the fundamental structural units of chromatin in metazoans (1, 2). The translational positioning of nucleosomes along DNA is implicated in profoundly influencing gene expression (3-6). Thus, defining the nucleosome positioning and occupancy is critical to understand the mechanisms of regulation of transcription by chromatin.
Nucleosome structure is resistant to microccocal nuclease (MNase) digestion, leaving a footprint of about 150bp that reflects the position of a nucleosome (7). Therefore, determining the boundaries of these footprints can indicate the positions of nucleosomes in the genome. Since the genomic sequences of most model organisms are already available, sequencing a short tag from DNA at each end of the nucleosome is sufficient to determine its position in the genome. Thus the next generation sequencing techniques are perfectly suited for this purpose (8).
We have generated genome-wide maps of nucleosome positions in both resting and activated human CD4+ T cells by direct sequencing of nucleosome ends using the Illumina Genome Analyzer Platform (MNase-Seq) (9). As the next generation sequencing techniques improve, the capacity and cost of sequencing become lower. For example, one sequencing run on the Illumina Genome Analyzer II can produce 100 to 200 millions of sequencing reads, which is sufficient to reach a 10x coverage for all nucleosomes in the human genome.
We describe two different methods to prepare nucleosome templates used for sequencing. One is digestion of native chromatin and the other is digestion of formaldehyde-crosslinked chromatin by MNase. The native nucleosome protocol works well to reveal stable nucleosome structure and avoid crosslinking of non-histone proteins; the crosslinking protocol may stabilize “unstable” nucleosomes but may also stabilize non-nucleosome structure that is resistant to MNase digestion.
Incubate the reaction mixture at room temperature for 45 min. Purify the DNA using QIAquick PCR Purification Kit. Elute DNA in 30 μl of EB (Note 5)
Incubate the reaction mixture at 70°C for 30 min. Purify the DNA as before. Elute DNA in 20 μl of EB (Note 5).
Incubate the reaction mixture at room temperature for 30 min.
Load 30 μl adaptor ligated DNA onto a 2% E-Gel EX (Invitrogen). Run 10min at EX Gel condition. Using E-Gel Safe Imager Real-time Transilluminator (Invitrogen), cut the gel around 200-400 bp region (DNA may not be visible). Extract the DNA using QIAquick Gel Extraction Kit(Note 4). Elute in ~23 μl EB (Note 5, Note 6).
Try 18 cycles first, check 2.5 μl of product on 1.8% gel. If the band is not clearly visible, do 3 more cycles. Check again.
Excise the band near 220 bp and purify the DNA using Qiagen gel extraction kit. Measure the DNA concentration using Qubit fluorometer (Note 4).
1Incubate the reaction for 8-10min at 37°C for formadehydecrosslinked nuclei.
2The E-Gel® agarose gel electrophoresis system is a complete bufferless system for agarose gel electrophoresis of DNA samples. It provides fast, safe, consistent, high-resolution electrophoresis and minimizes sample contamination. E-Gel® EX pre-cast agarose gels are generally used gels which contain a proprietary fluorescent nucleic acid stain with high sensitivity, allowing: (1)Detection of down to 1 ng/band of DNA, (2)Compatibility with blue light transillumination to dramatically reduce DNA damage, (3)Easy opening of cassette with gel knife. If this system is not available, traditional gel purification methods can be used, but cross-contamination may result.
3Unlike UV-transilluminators, the E-Gel® Safe ImagerTM Real-time Transilluminator does not produce UV light and does not require UV-protective equipment during use. Blue light transillumination also results in dramatically increased cloning efficiencies compared to UV transillumination.
4Dissolve gel slices at room temperature with frequent mixing, but not at elevated temperature. This helps to preserve AT-rich DNA that can be easily denatured at higher temperature and could then be lost at the column binding step.
5Warm up EB at 65°C. Ensure that the EB is dispensed directly onto the QlAquick membrane for complete elution of bound DNA.
6NEVER run PCR-amplified samples of Step 7 with the linker-ligated products of Step 5 together on the same gel because the latter can be contaminated.