High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

This method can answer key questions in the transcriptional regulation field, such as the role of chromatin interactions. The main advantage of this technique is that it provides a relatively unbiased capture of the interactions for a locus of interest. Although this method can provide insight into promoter enhancer interactions, it can also be applied to identify other types of chromatin interactions.

Generally, individuals new to this method will struggle because it takes several days for the protocol, and the primer design requires trial and error and atypical primer orientation. To preserve chromatin interactions in cells of choice, cross link them by adding 9.5 milliliters of 1%electron microscopy grade formaldehyde in PBS per 10 million cells. Incubate the cells while rocking for 10 minutes at room temperature.

Transfer the reaction tubes to ice to quench the cross linking reaction and add ice cold one molar glycine to a final concentration of 0.125 molar and mix by gentle inversion. After centrifuging and washing the cells according to the text protocol, re suspend the pellet in 125 microliters of five millimolar EDTA with 0.5%SDS and one X protease inhibitors. Incubate the cell suspension on ice for 10 minutes.

To make sure that the cell lyse is complete, mix six microliters of the cells with six microliters of trypan blue on a microscope slide and cover with a cover slip. View under a microscope to see the interior of the lysed cells blue, and unlysed cells white. For the first restriction digestion add 30 microliters of 10 X restriction enzyme buffer, and 27 microliters of 20%Triton X-100 to the cell suspension and adjust the total volume to 300 microliters with water.

Remove a 15 microliter aliquot and store at four degrees Celsius as undigested control. To the remaining reaction mixture add 200 unites of restriction enzyme one. Incubate overnight at the temperature appropriate for the enzyme in a shaking heating block with 900 RPM agitation.

The following day, add an additional 200 units of the enzyme and continue this incubation overnight. Remove a 15 microliter aliquot and store at four degrees Celsius as digested control. To determine digestion efficiency add 82.5 microliters of 10 millimolar Tris-HCl, pH 7.5, to each undigested and digested controls.

Add 2.5 microliters of proteinase K and incubate for one hour at 65 degrees Celsius to reverse the formaldehyde cross linking. To isolate the digested DNA, add 100 microliters of phenol chloroform to the tube. Mix by several quick inversions to remove residual protein contamination.

Centrifuge at 16, 100 G's at room temperature for five minutes. After centrifugation, transfer the aqueous phase to a new tube. Add sodium acetate, glycogen, and 100%ethanol to the tube and mix gently by inversion.

Incubate at minus 80 degrees Celsius for one hour to precipitate the DNA. Centrifuge the tube at 16, 100 G's at four degrees Celsius for 20 minutes. Remove the supernatant, and then add 500 microliters of 70%ethanol to wash the DNA pellet.

Centrifuge at 16, 100 G's at room temperature for five minutes. After removing the supernatant air dry the pellet at room temperature for two minutes to remove residual ethanol. Re suspend the dried pellet in 50 microliters nuclease free water and proceed to determine digestion efficiency by QPCR as described in the text protocol.

To prepare for ligation, heat and activate the restriction enzyme by incubating the tube for 20 minutes at 65 degrees Celsius. Then transfer the contents of the tube to a 50 milliliter conical tube, and add nuclease free water, 10 X ligase buffer, and T4 DNA ligase. Mix gently by swirling, and incubate overnight at 16 degrees Celsius and proceed as described in the text protocol.

To reverse cross linking add 15 microliters of proteinase K and incubate overnight at 65 degrees Celsius. On the following day add 30 microliters of RNase A, and incubate at 45 minutes at 37 degrees Celsius. To continue with chromatin isolation, add seven milliliters of phenol chloroform and mix by several quick inversions.

Centrifuge the tube at 3, 300 G's at room temperature for 15 minutes. After centrifugation, transfer the aqueous phase to a new 50 milliliter tube, and add nuclease free water, three molar sodium acetate, glycogen, and 100%ethanol. Mix the contents and incubate at negative 80 degrees Celsius for one hour.

After incubation, centrifuge the tube at 3, 900 G's at four degrees Celsius for 20 minutes. Remove the supernatant, and then wash the pellet with 10 milliliters of ice cold 70%ethanol. Centrifuge at 3, 300 G's at four degrees Celsius for 15 minutes.

Remove the supernatant, and briefly dry the pellet at room temperature. Dissolve the pellet in 150 microliters of 10 millimolar Tris-HCl pH 7.5 at 37 degrees Celsius. Store at negative 20 degrees Celsius or continue with second restriction digestion, ligation, and DNA purification as described in the text protocol.

Following DNA purification, perform QPCR using inverse PCR primers and cyber and ROX dyes to determine the number of amplification cycles for inverse PCR amplification of unknown interacting sequences as described in the text. To prepare DNA for sequencing, trim off bait sequences with third restriction digestion by digesting one microgram of purified product of inverse PCR as well as restriction enzyme monitor. Run the digested restriction enzyme, or RE monitor, on an agarose gel of a concentration appropriate for the expected fragments.

Once digestion efficiency has been determined by gel electrophoresis, continue with inverse PCR product purification and preparation of sequencing library, as described in the text protocol. Third restriction digestion is important in this protocol for next generation sequencing of circular chromosome confirmation capture. This digestion trims off bait sequences which can facilitate the identification of chromatin attractions.

Agarose gel electrophoresis of digested RE monitor indicated sufficient digestion of the inverse PCR product digested in parallel. After finished four C sequencing reads were trimmed and mapped to human reference genome hg38, using BWA software package. The majority of reads align at restriction sites HindiIII or CviQ1I sites adjacent to HindiIII sites as expected.

While attempting this procedure it's important to make sure your cells are lysed and the chromatin is sufficiently digested. Following this procedure, other methods like chromatin immuno precipitation or CHIP, can be performed in order to answer additional questions such as identifying transcription factors involved in the chromatin interactions. After its development, this technique paved the way for researchers in the chromatin field to explore physical regulatory networks.