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.
You can optimize the library prep protocol to reduce the amount of DNA required. For some applications, such as ChIP-Seq, where DNA amounts might be low, you can skip steps such as size selection on a gel. Instead, end-repaired DNA fragments can be ligated, purified by a column cleanup step that removes primer-dimers, and then quantitated and directly loaded onto the flow cell for cluster amplification.
For genomic DNA (gDNA) from small samples, optimize fragmentation to increase the number of DNA fragments that are within the target size range. If these fragments are within a tight size range, you can eliminate the agarose gel step.
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.
Illumina’s proprietary method ensures ligation of 2 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.
Common controls include no primary antibody or use of normal rabbit IgG in ChIP as a negative control. In most cases where you are studying the same factor in different tissues or cell lines, one control is sufficient. More controls are needed when 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 control is typically generated by the following process: After sonication, divide the sample into two equal parts and add the antibody to one part and a mock antibody (normal IgG) or preimmune serum to the other.
The goal is to make sure that you are not seeing sequences that stick nonspecifically to the antibodies and that no other part of the process is influencing the results (a process control). Additionally, it ensures that your antibody recognizes your protien as a requirement for enrichment of the target sequence.
Illumina has received feedback from several successful ChIP-Seq researchers who have said that a mock control is the best, with the lowest background, but does require more work than an input control.
See the standard protocols for the resequencing genomic DNA.
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. The concatemers 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 20x or 50x dilution, rather than the 10x dilution described in the protocol).
For studies targeting transcription factors, Illumina recommends 5–15
M 1×35–1×50 reads per sample. For studies targeting histone
modifications, we recommend 50–90M 1×35–1×50 reads.
Sequence ChIP-Seq libraries prepared with the ChIP-Seq library prep kit on a single-read flow cell. Libraries prepared with paired-end adapters can be run on either single-read or paired-end flow cells.