Since regulatory elements such as transcriptional enhancers can be located at large genomic distances from their target genes which they regulate through long range chromosomal interactions, the goal of Promoter Capture Hi-C is to link gene regulatory elements to their target genes. This method can help answer key questions in the field of genome structure and function such as how the three dimensional architecture of a genome affects gene expression control. The main advantage of this technique is that it allows the detection of significant promoter interactions, a single restriction from the solution.
The implications of this technique extend towards therapy because it can link noncoding disease associative sequence variants in or near regulatory control sequences to their target genes. Start with a minimum of 20 million cells per experiment. For suspension cells collect and centrifuge cells at 400 times g and 20 degrees celsius for three minutes.
Remove the supernatant and re-suspend the cell pellet in 30.625 milliliters of medium with 10%FBS at room temperature. Then add 4.375 milliliters of 16%methanol-free paraformaldehyde to a final concentration of 2%Fix the cells for 10 minutes at room temperature with gentle mixing on a rocker. Quench the reaction by adding 5 milliliters of freshly prepared one molar ice-cold glycine.
Mix for five minutes with gentle rocking at room temperature. Then incubate on ice for 15 minutes with occasional inverting. Next centrifuge the suspension cells at 760 times g and four degrees celsius for five minutes.
Remove the supernatant and re-suspend the cell pellet in 50 milliliters of ice cold PBS pH 7.4. Finally centrifuge the cells at 400 times g and four degrees celsius for 10 minutes and carefully remove the supernatant. Following cell lysis as described in the text protocol wash the cell nuclei with 1.25x restriction buffer 2.
To do so re-suspend the pellet in one milliliter of ice cold buffer and transfer the re-suspended nuclei into a 1.5 milliliter tube. Spin the nuclei at 760 times g and four degrees celsius for five minutes before removing the supernatant. Re-suspend the pellet in 1790 microliters of 1.25x restriction buffer 2.
Then divide the suspension into five aliquots each containing five to 10 million cell nuclei in 358 microliters. Add 11 microliters of 10%weight per volume SDS per aliquot and shake at 950 revolutions per minute for 30 minutes at 37 degrees celsius in a thermomixer. If cell clumps appear dissociate them by pipetting avoiding making bubbles.
Next add 75 microliters of 10%Triton X-100 per aliquot and shake at 950 rpm and 37 degrees celsius for 15 minutes in a thermomixer. Add 12 microliters of 100 units per microliter of HindIII per aliquot. Incubate at 37 degrees celsius overnight while shaking at 950 rpm in a thermomixer.
Following digestion perform quantitative PCR as detailed in the text protocol. Next add 60 microliters of biotinylation master mix per aliquot and mix. Incubate the aliquots at 37 degrees celsius for one hour shaking at 700 rpm for five seconds every 30 seconds.
After one hour place the aliquots on ice. Perform the Hi-C library as detailed in the text protocol. Transfer between 500 nanograms and one microgram of the Hi-C library into a new tube then evaporate the sample on a vacuum concentrator until dry.
Re-suspend the evaporated Hi-C library by adding 3.6 microliters of molecular biology grade water, 2.5 microliters of blocker 1 2.5 microliters of blocker 2 and 0.6 microliters of custom blocker. Transfer the sample into a well of a new PCR tube strip close it with the PCR cap strip and place the sample on ice. Label the well as D for Hi-C DNA.
Prepare the hybridization buffer as described in the text protocol. Incubate the buffer at 65 degrees celsius for five minutes in a thermomixer. Transfer the buffer into a well of a new PCR tube strip close it with the PCR cap strip and keep at room temperature.
Label the well as H for hybridization buffer. Into a well of a new PCR tube strip mix five microliters of 100 nanogram per microliter biotinylated RNA probes, 0.5 microliters of SRNase B RNase inhibitor, and 1.5 microliters of molecular biology grade water. Close the PCR tube strip with a PCR cap strip and place on ice.
Label the tube as R for RNA. Set up the PCR machine as detailed in the text protocol. Proceed as quickly as possible during all procedures while the PCR machine is running in order to avoid sample evaporation.
Place the D PCR tube strip in the PCR machine close the PCR machine lid and start the PCR reaction. When the PCR program reaches 65 degrees celsius open the PCR machine lid and place the H PCR tube strip in the PCR machine. Close the PCR machine lid and incubate for three minutes.
Then open the PCR machine lid place the R PCR tube strip in the PCR machine and close the PCR machine. After two minutes open the PCR machine lid and all PCR tube strips. Transfer 13 microliters of well H into well R and then all volume of well D into well R.Pipet up and down three times to mix the reaction and close the PCR tube strip.
Remove the H and D PCR tube strips and close the PCR machine lid. Incubate the reaction at 65 degrees celsius for 24 hours. Add 60 microliters of streptavidin-coupled magnetic beads into a new tube.
Place the tube on the magnetic separation stand for one minute before removing the supernatant. Re-suspend the beads with 200 microliters of 1x binding buffer and rotate for three minutes at room temperature and 15 rpm on a rotating wheel. Softly spin the tube in a benchtop centrifuge for two to three seconds to collect the sample.
After placing the tube on the magnetic separation stand for three minutes remove the supernatant. Repeat this wash twice. Next re-suspend the beads in 200 microliters of 1x binding buffer.
Open the PCR machine and the PCR tube strip while the PCR program is still running and transfer the hybridization reaction into the tube with the magnetic beads. Incubate the reaction and beads at room temperature for 30 minutes on a rotating wheel at three rpm. Reclaim the beads on the magnetic separation stand and remove the clear supernatant.
After re-suspending the beads in 500 microliters of wash buffer 1 incubate the sample for 15 minutes at 20 degrees celsius while shaking at 950 rpm in a thermomixer. Again reclaim the beads and remove the clear supernatant. Now re-suspend the beads in 500 microliters of wash buffer 2 and mix.
Incubate the sample for 10 minutes at 65 degrees celsius while shaking at 950 rpm in a thermomixer. Repeat this wash with wash buffer 2 twice more. Then place the sample onto the magnetic separation stand remove the clear supernatant and re-suspend the beads in 200 microliters of 1x restriction buffer 2.
After reclaiming the beads and removing the supernatant again re-suspend the beads into 30 microliters of 1x restriction buffer 2. This figure shows the promoter capture Hi-C interaction profile for the IL8 promoter in 17 human hematopoietic cell types. Promoter interactions of lymphoid cell types are shown as purplearches and promoter interruptions of myeloid cell types are shown as blue arches.
Monocyte specific interactions are indicated by green arrows neutrophil specific interactions are indicated by red arrows and a megakaryocytes specific interaction is indicated by a brown arrow. While attempting this procedure it's important to remember to work as precisely as you can. It's not black magic but it is a lengthy protocol so small mistakes will add up to negatively affect data quality in the end.
Following this procedure other methods like CRISPR/Cas9 genomore epigenome editing can be performed in order to interrogate the biological function of promoter interacting regions uncovered by promoter Capture Hi-C. After watching this video we hope you would have a good understanding of how to implement promoter Capture Hi-C in your lab.
