Questions & Answers

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General

  • How is the TruSeq ChIP Sample Prep kit different from the legacy ChIP-Seq Sample Prep kit?

    The TruSeq ChIP Sample Prep kit generates Paired End (PE), indexed libraries that are compatible with all Illumina sequencing platforms and can be multiplexed. The legacy ChIP-Seq Sample Prep kit generates Single Read (SR) and non-indexed libraries that are compatible with Genome Analyzer, HiSeq and HiScanSQ, but not MiSeq.

Input

  • How much starting material does the ChIP-Seq Sample Preparation Kit require?

    The ChIP-Seq Sample Preparation Kit is designed to use 10 ng of ChIP-enriched DNA. Many users have reported that using at least 30 ng of DNA makes the protocol simpler and more repeatable.

  • What are Illumina's recommendations for preparation of ChIP DNA?

    We do not have any specific recommendations regarding the chromatin immunoprecipitation step, as techniques for this process can vary widely based on the desired application. However, we do not recommend using any type of nucleic acid as a carrier in the chromatin immunoprecipitation prior to sequencing. The carrier nucleic acid is difficult to remove from the sample, and can end up being 50–90% of the final library.

  • Can the ChIP-Seq protocol be used to make libraries from low starting amounts of non-ChIP DNA?

    Yes, although this requires some adaptation of the protocol. The ChIP-Seq protocol assumes that the input DNA is already fragmented. If the intended starting material is genomic DNA, it will need to be fragmented before use with this protocol. Fragmentation methods such as Covaris offer higher recovery and a tighter size distribution than nebulization, and are preferred for this sort of application. At least 10 ng fragmented DNA should be used.

  • What if I don't have enough template DNA?

    The library process can be optimized to reduce the amount of DNA required. For some applications such as ChIP-Seq where DNA amounts might be low, steps such as size selection on a gel can be skipped. Instead, end-repaired DNA fragments can be ligated, purified by a column clean-up step that removes primer-dimers, and then quantitated and directly introduced to a flow cell for cluster amplification. For genomic DNA from small samples, fragmentation can be optimized to increase the number of DNA fragments that are within the target size range. If these DNA fragments are within a tight size range, then the agarose gel step can be eliminated.

Protocol

  • How should ChIP DNA be quantitated?

    We recommend quantitating ChIP DNA with the most sensitive method available, taking into account the low amount of DNA that is often available. Fluorescent dye-binding assays such as PicoGreen or Qubit offer high accuracy, assuming precise pipetting. The Bioanalyzer and High Sensitivity chips can also give good quantitation, and allow you to check the size and purity of your sample.

  • How do you ensure that different adapters are ligated to each end of a DNA fragment using this protocol?

    Illumina’s proprietary method ensures ligation of two different adapters in the required orientation to opposing ends of a DNA fragment. PCR selects for these and finalizes the construct ready for hybridizing onto the flow cell surface. Adapter sequences can be determined by sequencing the ligation fragments, but sequence information alone is not sufficient to uncover the method.

  • What types of controls should be run for a ChIP assay?

    Common controls include no primary antibody or use of normal rabbit IgG in ChIP as a negative control. In most cases, if you are studying the same factor in different tissues or cell lines, one control is sufficient. More controls will be needed if you are studying multiple factors from the same source.

    We have seen researchers use either a genomic DNA input control or a "mock" control with IgG. A mock sample is usually generated by the following process: after sonication, divide your sample into two equal parts and add your antibody to one part, while adding mock antibody (normal IgG) or pre-immune serum to the other. The goal is to ensure that you are not seeing sequences that stick non-specifically to the antibodies and that no other part of the process is influencing your results (a process control). In addition, it ensures that the recognition of your protein by the antibody you are using is required for enrichment of the target sequence. We have received feedback from several successful ChIP-Seq researchers who have told us that a mock control is the best, with the lowest background, but this does require more work than an input control.

  • What should I do if there is an issue with the Seq part of my ChIP-Seq?

    Please refer to the standard protocols for the resequencing of genomic DNA.

Library Evaluation

  • I see a 130-135 bp band on my final ChIP-Seq library. What is this?

    Although the Illumina adapters are designed to reduce adapter-adapter ligation, very low amounts of starting material can result in high adapter-insert ratios during ligation. This promotes formation of adapter dimers and occasional adapter concatamers. These concatamers often take the form of amplified trimers in various configurations. They can be removed by careful size selection, or by repeating the ligation with more input DNA or less adapter. The amount of adapter added to the ligation can be titrated by additional dilution (i.e. a 20× or 50× dilution, rather than the 10× dilution described in the protocol).

Cluster Generation

  • What cluster generation kit should be used for this sample prep library?

    For kit compatibility information, see the Illumina Version Compatibility Reference.

  • Can pre-TruSeq libraries be run on TruSeq cluster kits?

    Yes, TruSeq cluster kits are backwards-compatible.

Sequencing

  • What is the recommended minimum number of reads per sample and sequencing format for ChIP-Seq?

    For studies targeting transcription factors, we recommend between 5 and 15M 1×35–1×50 reads per sample. For studies targeting histone modifications, we would recommend between 50–90M 1×35–1×50 reads. 

  • What flow cell should be used with ChIP-Seq libraries?

    ChIP-Seq libraries constructed with the ChIP-Seq sample preparation kit should be run on single-read flow cells. Libraries constructed with paired-end adapters can be run on either single-read or paired-end flow cells.

Analysis

  • Are there third-party software programs that you can recommend for ChIP-Seq analysis?

    There are a number of solutions available, including Bioconductor and MACS, which are available through Galaxy.