Generating a library of DHSs
The basic protocol is outlined in Figure 1. Nuclei are extracted from living cells, then digested on ice for 1 h with a range of DNaseI concentrations, as detailed in the ‘Materials and methods’ sections. Following RNaseA and proteinaseK treatment, the DNA is extracted and run on a 1% agarose gel to check for the size of digested DNA. The gel in Figure 1A shows the DNA from mouse thymocyte nuclei digested with 0, 40 and 120 units of DNaseI. Maximal enrichment at DHS is usually observed with samples that are not over-digested. In the experiment shown, maximal enrichment at a known control DHS was seen with 40 units DNaseI (real-time PCR quantification shown in Figure 2B). The DNA is then blunt-ended using T4 polymerase (Figure 1B) and ligated with an asymmetric double-stranded linker. After extraction, DNA is amplified using a biotinylated linker-specific primer and 35 thermal polymerase cycles, as detailed in ‘Materials and methods’ section. As the linker will ligate to both ends of digested DNA, the amplification will represent a mix of primer extension and PCR, depending on the length of DNA amplified. As has been previously reasoned (13), one double strand of DNA in a region of DNaseI hypersensitivity is more likely to be digested twice within a short distance than non-hypersensitive DNA. This will lead to the preferential amplification of DNA from regions of DHS. The amplified DNA is then extracted using para-magnetic streptavidin beads, which provides a DNA library representative of whole-genome DHS. Agarose gel electrophoresis of the DNA recovered from the beads confirms that the vast majority of these products are between 300 and 500 base pairs in length (Figure 1B).
Figure 1. (A) Nuclei are isolated from cells, and aliquots are digested with a range of DNaseI as detailed in ‘Materials and methods’ section. The DNA is extracted and run on 1% agarose gels using gel electrophoresis (right panel). With the example shown, the 40-units sample gave maximal enrichment at a housekeeping promoter using the complete protocol. (B) The digested DNA is blunt-ended with T4 polymerase and ligated to the LP21–25 linker as detailed in ‘Materials and methods’ section. Following amplification with the biotinylated LP25 primer, the extracted DNA template represents a library of whole-genome DHS. When samples of this library are visualised using gel electrophoresis (bottom panel), the majority of products are between 300 and 500 bp in size.
Figure 2. (A) The left-hand panels show the Sybr-green real-time PCR profiles (fluorescence versus cycle number) of 5-fold dilutions of quantified mouse genomic DNA standards for two primer pairs within (ppA) and 3′ (ppB) of the Stil promoter. The right-hand panel shows that DNA template derived from the 40 units DNaseI-treated sample amplified nearly six PCR cycles in advance of the 0 DNaseI-treated sample with ppA. No difference in amplification kinetics was seen between the 0 and 40 samples with ppB. (B) Quantification of samples relative to genomic standards using primers for the ‘housekeeping’ Hmbs promoter shows maximal enrichment with 40 units DNaseI treatment for primary mouse thymocytes (left panel) compared with maximal enrichment with 120 units DNaseI for the mouse T-cell line, BW5147 (right panel).