Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

The overall goal of this procedure is to provide researchers with an automated platform capable of performing chromatin immunoprecipitation experiments on just 10, 000 cells. This is accomplished by first preparing high quality sheared chromatin for the chromatin immunoprecipitation. In the second step, an automated chromatin immunoprecipitation experiment is created and automated libraries are run with the immuno precipitated DNA.

The immuno precipitated DNA is analyzed by quantitative PCR and next generation sequencing. Ultimately, the success of the automated technology can be validated through the comparison of the chromatin immunoprecipitation sequence profiles generated on the original 10, 000 cell sample with experiments performed on 100, 000 cells and existing data sets for reference. This method can help to answer key questions in the epigenetic field to further our understanding of the functions of specific biomarkers in various tissues.

Stages of development and disease states. This technique has implications toward the diagnosis of multiple diseases as it will help to identify epigenetic biomarkers in health and disease, Accurate and reliable. Automatic technologies are important tools for researchers in epigenetic analysis of specific cell types, subpopulations and biopsies.

For a standard automated chromatin immunoprecipitation experiment begin by adding 120 microliters of chromatin immunoprecipitation buffer H to 100 microliters of sheared chromatin from one to 2 million cells, reserving two to 20 microliters of chromatin for the input sample. Then to set up the automated experiment on the IP star compact select protocols and click on the chromatin immunoprecipitation icon. Choose the chromatin immunoprecipitation direct method, and then in the next screen, choose the chromatin immunoprecipitation 200 microliter protocol and specify the sample number.

Set the chromatin immunoprecipitation experimental parameters to four hours for the antibody coating. Step 15 hours for the immunoprecipitation step, and five minutes for each washing step. Then set up the reagents following the instructions displayed on the layout information screen, and add the appropriate reagents and highly validated ChIP-seq grade antibodies.

Then add 20 microliters of protein, A coated magnetic beads for each reaction, and the chromatin samples after the overnight chip protocol and the reverse cross-linking are over. Purify the DNA with a magnetic bead based DNA purification kit. Use a highly sensitive commercial fluoro metric assay to quantify the immuno precipitated DNA.

Next, using primers for at least one positive and one negative control genomic region. Analyze the quality of the immuno precipitated DNA by quantitative PCR according to the appropriate parameters of the materials for the analysis. The results are expressed as the percentage of the recovery of the input for the next generation sequencing under protocols.

Now click on the library prep icon, then choose the library prep technology of preference and set up the reagents according to the instructions on the layout information screen. For example, in this experiment, sequencing profiles for six different histone modifications associated with gene expression were generated. The high peak correlation observed between these histone markers indicates the ability of the automated system to generate accurate and reliable data.

Moreover, these graphs show the distribution of the peaks for the different histone modifications at specific genomic regions. Finally, for an automated chromatin immunoprecipitation experiment for samples with low cell numbers, set the automated chromatin immunoprecipitation 200 microliter protocol on the automation system as just demonstrated. Next, load the system with the ChIP-seq, grade antibodies and reagents optimize to work on chromatin quantities between 10, 000 and 100, 000 cells using only 10 microliters of protein, A coated magnetic beads for each reaction.

After chip, run the library preparation, select the micro plex library prep protocol and set up the reagents following the instructions displayed on the layout information screen. In this representative quantitative PCR of a chromatin immunoprecipitation from a 10, 000 cells starting sample, significant enrichments with the anti histone antibodies in the positive control regions and negligible signals in the negative control regions were observed. Here a series of 10 immunoprecipitation reactions that were reproducible and highly comparable with a manual chromatin immunoprecipitation experiment are shown demonstrating the ability of the automated system to work with low cell quantities with no visible increase in experiment to experiment variability compared to manual immunoprecipitation.

These data illustrate the bioinformatics analyses of the sequence libraries of 10, 000 and 100, 000. He a S3 cells after chromatin immunoprecipitation, demonstrating outstanding results from the low cell number chromatin immunoprecipitation samples. This dataset, which corresponds to the experiment with 10, 000 cells of starting material, contains low background noise with highly reliable enrichment peaks that are confirmed by both the 100, 000 cell dataset and the Broad Institute reference dataset.

For the encode project, it is important to note that the 10, 000 cell data demonstrate almost identical peaks to the Broad Institute data with a 98%overlap ratio of the best 40%of the peaks ranked by the significant score as used in the end code project. The IP star automated system requires minimal operator intervention, reducing the HandsOn time of the chromatin immunoprecipitation to just one to two hours. In addition, the automated process replaces the numerous error prone steps of the manual chromatin immunoprecipitation, and delivers highly consistent results.

While attempting this procedure, it's important to remember that the good chromatin immunoprecipitation and a good selection of highly specific and sensitive antibodies is key for the success of the experiment. This technology paved the way for researchers in every field of biology to improve the accuracy, reproducibility, and consistency of therapy. Epigenetics studies.