First things first, you’re probably wondering how long this is going to take. To read this post, probably only a few minutes, but the full HiChIP assay process will take approximately 2.5 days from sample to having your libraries in-hand. In the figure below, we break down the stages and approximate time demands for each stage. The stages in the green box are what you’ll do on day one, with day two in the orange box and day three in the blue box.
Now, let’s dive into the workflow. During day one (steps outlined in green), the very first thing you’ll do is crosslink the chromatin in the nucleus (in situ). This will preserve the native chromatin architecture as you progress through the protocol. Once the crosslinking step is done, you’ll use micrococcal nuclease (MNase) to digest the chromatin. MNase is unusual in that it possesses both endonuclease and exonuclease activity. This results in consistent breaks at the edges of the nucleosome protected DNA resulting in fragments representative of nucleosome position and provides nucleosome-resolution results. After the DNA has been digested, your cells are lysed, the sample is spun down, and the supernatant containing your chromatin fragments is incubated overnight with your antibody of interest. Pretty straightforward first day!
Day two (steps outlined in orange) starts with immunoprecipitation, similar to ChIP-seq, followed by proximity ligation reactions. For those unfamiliar with ChIP, DNA-protein-antibody complexes are pulled down with protein A/G paramagnetic beads. Subsequently, the precipitated material is subjected to proximity ligation, and finally the crosslinks are reversed and the DNA is purified.
At this point, you’ll want to check how much DNA you recover, as this will be your library preparation input. A QC step is included in the protocol to quantify your DNA yield (last step of stage 4). A high yield at this stage predicts a high complexity library, so it’s a good sign if you pull down a lot of DNA molecules that have been anchored with your protein of interest. Phew, that’s a wrap on day two!
On your third and final day (steps outlined in blue), you’ll convert the proximity ligated DNA molecules into sequence-able molecules, which is ultimately what we’re pursuing. Prior to PCR amplification, you will perform a second pull-down- this time using streptavidin beads- to enrich for the biotinylated proximity ligated DNA products. Last but certainly not least, you’ll amplify those molecules by PCR, and from there go on to sequencing.
Now that you have the gist of *what* to do, we figured it would be helpful to address some of the common questions we hear…
Historically, sonication has been used to fragment DNA. However, we’ve found that enzymatic digestion using MNase provides a more consistent, uniform digestion profile, delivering the highest resolution view of chromatin architecture, excellent library complexity, and a high percentage of long-range interactions.
Ideally, you want your digested chromatin to contain between 40-70% mononucleosomes. Having less than 40% mononucleosomes generally means the chromatin has been under-digested, and more than 70% is an over-digestion. Under or over-digestion typically results in lower lysate yield and lower complexity, so you might see a high duplication rate in the downstream analysis.
In stage 2 of the protocol, following RIPA cell lysis, we ask that you to do a quick QC check to assess your digestion profile. Using an aliquot of the clarified lysate, you will reverse the crosslinks, treat with proteinase K, purify the DNA, and then analyze on a Fragment Analyzer, Bioanalyzer, or TapeStation to assess the fragmentation size distribution. This ensures that time spent creating your library will yield the best library possible.
The User Manual includes a troubleshooting guide that makes adjusting the digestion easy. Our technical support team is also available to help if necessary.
The ratio between your chromatin lysate and antibody is very important for ensuring a successful HiChIP experiment (no different from running a ChIP-seq assay). Dovetail has validated a large panel of the most commonly used antibodies (including histone modifiers and transcription factors) and guidance for the use of each antibody is provided in the User Guide.