Hi David!
I'm glad you used this protocol for your experiment. And I'm sorry for answering late. Based on your questions, I will provide answers below, hoping they can be of assistance to you.
- We started the experiment by using RNAseZap to wipe down the entire laboratory bench and the hood. We didn't perform any of the pre-reverse transcription steps under a hood, but recently we detected that incubated on ice for 5mins after adding SuperScriptTM IV RT buffer and SuperScriptTM IV reverse transcriptase will slightly improve the efficiency of reverse transcription.
- Before FACS, we used the thermomixer throughout the entire process. Reverse transcription: 55°C for 15 mins. RE digest: overnight (about 12 hours). Ligation: 16°C for 4 hours
- The difference between MboI and DpnI is solely in their sensitivity to methylation. The choice of MboI is because we are not concerned with the methylation status.
- Typically, adding 1% Triton X-100 during the ligation step is to further permeabilize the cell nuclei, allowing enzymes to enter adequately. Another function is to facilitate subsequent centrifugation steps, much like in standard Hi-C experiments where cells are collected by centrifugation. We believe that our previous steps have sufficiently permeabilized the cell nuclei, and we do not require centrifugation. Following that, we proceed to sort cells using FACS, hence we have omitted the addition of extra Triton X-100.
- We discovered a phenomenon where there is a delicate balance between Hi-C and transcription, such that adding an excess of transcriptase reduces the Hi-C contacts, and the reverse is also true. This will require you to make some minor adjustments based on your experimental needs.
- We did not attempt overnight ligation because it coincided with the subsequent FACS cell sorting. However, you can try to observe whether overnight ligation will lead to fragment growth based on the results of the FA plot. Generally, we believe that the longer the ligation, the better the effect.
Hope these answers help you.