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…