Diagenode's High Resolution Library Preparation Kit significantly enhances the resolution of transcription factor binding sites to single nucleotide levels. Using an optimized, proprietary protocol using an exonuclease to digest protein-bound DNA from the 5' end to within a few nucleotides of the protein binding site, the precise location of transcription factor-bound DNA can be determined. The High Resolution Library Preparation Kit provides a much higher resolution than in ChIP-seq and thus more accurately determines binding sites.
The High Resolution ChIP-seq Library Prep kit is the only kit available allowing the mapping of transcription factors using exonuclease. With this kit, we were able to determine very precisely the bound DNA motifs of FoxA1 at a genome-wide scale. Moreover the signal/background ratio is about 3-fold higher compared to traditional ChIP-seq which means that this kit may also improve signal from sub-optimal antibodies.
Dr. Aurelien Serandour, Lab of Dr. Jason Carroll, University of Cambridge
Figure 1: After ChIP, a P7-adaptor is ligated to the ChIP'd DNA. Then a 5-to-3 lambda exonuclease digests up to the transcription factor binding site and selectively eliminates the P7 adaptor sequence attached at the 5 end of each strand. After crosslink reversal and elution the single-stranded DNA is made doublestranded by P7 PCR primer extension. Afterwards a P5 adaptor is ligated to the exonuclease-treated end, and the resulting library anlyzed by Next- Generation Sequencing By mapping the 5 ends of the resulting sequencing tags to the reference genome the bound motif can be determined accurately.
Figure 2: Two ChIP-seq experiments were performed using an antibody against ER alpha on the same samples. The libraries for one of the experiments (top lane, red) were produced using the High Resolution Library Preparation Kit, the other (bottom lane, blue) is a standard ChIP-seq experiment. The figure shows the enrichments near the known ER alpha target gene ABCA3 as a bar chart of read coverage. The scale of both profiles is identical. Due to the tighter read distribution, the High Resolution Library Preparation Kit enables the accurate identification of the actual binding
Figure 3: Two ChIP-seq experiments were performed using an antibody against ER alpha on the same samples. The libraries for one of the experiments (top lane, red) were produced using the High Resolution Library Preparation Kit, the other (bottom lane, blue) is a standard ChIP-seq experiment. The figure shows the enrichments near the known ER alpha target gene GREB1 as a bar chart of read coverage. The scale of both profiles is identical. It is evident that with the High Resolution Library Preparation Kit the read distribution is significantly tighter around the binding sites, giving a higher enrichment and less background.
