Our research aims to uncover how chromatin organization controls gene expression. The focus is on determining what factors regulate chromatin at the gene level to control gene activity. Currently, technology similar to chromosome confirmation capture or 3C are used to make progress in this field.
Advancements in sample collection and sequencing technologies have pushed us what the 3C techniques can show us. The greatest experimental challenge currently is collecting samples having uniform chromatin architecture. Heterogeneity in samples leads to confounding results.
In the future, we'll be focusing on how changes in chromatin organization play a role in cancer. On day one, after chromatin cross-linking and nuclei collection from 5, 000 young adult Caenorhabditis elegans worms, transfer the nuclei pellet resuspended in 450 microliters of clean water to a clean 1.5 milliliter micro centrifuge tube. Then add 60 microliters of 10x DpnII restriction enzyme buffer to it and mix well.
Incubate the samples with 15 microliters of 10%sodium dodecyl sulfate at 37 degrees Celsius for one hour with agitation. Quench the reaction by adding 75 microliters of 20%Triton X-100 and incubating as demonstrated previously. Aliquot 10 microliters from the sample as the undigested control and store it at four degrees Celsius.
After adding 400 units of DpnII to the rest of the sample, incubate it overnight at 37 degrees Celsius with agitation for chromatin digestion. On day two, add an additional 200 units of DpnII to the sample. To complete the digestion of the cross-linked sample, incubate it at 37 degrees Celsius with agitation for four hours.
Heat inactivate the restriction enzyme by incubating the sample for 20 minutes at 65 degrees Celsius. If the enzyme cannot be inactivated by heat, add 80 microliters of 10%sodium dodecyl sulfate and incubate. Then add 375 microliters of 20%Triton X-100 to the sample and mix by swirling.
Keep aside a 10 microliter aliquot from the sample as the digestion control. For chromatin ligation, transfer the sample to a clean 50 milliliter conical tube. Adjust the sample volume to 5.7 milliliters with molecular grade water and mix by swirling.
Then add 700 microliters of 10x T4 ligase buffer, followed by 60 units of T4 DNA ligase, and incubate the sample overnight at 16 degrees Celsius. On day three, proceed with protein digestion and reverse cross-linking. Add 30 microliters of 10 milligrams per milliliter proteinase K to the 3C sample and incubate at 65 degrees Celsius overnight with agitation.
To begin purification of the 3C library on day four, add 30 microliters of 10 milligrams per milliliter RNase A to the sample and mix by swirling. After incubation, add seven milliliters of phenyl chloroform to the samples and mix by shaking. Centrifuge the sample for 15 minutes at 3, 270 G and room temperature.
Collect and transfer the aqueous phase to a clean 50 milliliter conical tube. Add equal volumes of chloroform and mix the sample by shaking. After centrifuging the sample again as demonstrated, transfer the aqueous phase to another clean 50 milliliter conical tube.
Add 7.5 milliliters of molecular grade water, 35 milliliters of 100%ethanol and seven microliters of one milligram per milliliter glycogen. Freeze the properly mixed sample at minus 80 degrees Celsius. While the sample freezes, begin purifying the control samples by adjusting the volume of the controls to 500 microliters using molecular grade water.
Add two microliters of 10 milligrams per milliliter RNase A and mix by flicking the tube. Next, add one milliliter of phenyl chloroform to the tubes containing the controls and mix by shaking. Centrifuge the tubes.
After transferring the aqueous phase to a clean 1.5 milliliter micro centrifuge tube, add equal volumes of chloroform. Centrifuge as previously demonstrated before collecting the aqueous phase. To the collected aqueous phase, add one milliliter of ethanol and two microliters of one milligram per milliliter glycogen.
After mixing the sample by shaking, incubate it at minus 80 degrees Celsius for 30 minutes. Next, centrifuge the sample for 15 minutes at 3, 270 G at four degrees Celsius. After removing the supernatant, add 750 microliters of chilled 70%ethanol and centrifuge.
Resuspend the air dried pellet in 50 microliters of molecular grade water. The suspension can be frozen here, if not proceeding further immediately. To continue with the 3C sample purification, remove the sample from the freezer and centrifuge at 3, 270 G for 30 minutes.
Add 10 milliliters of chilled 70%ethanol and break up the DNA pellet. After centrifuging and removing the supernatant, partially air dry the sample at room temperature. Resuspend the pellet in 150 microliters of 10 millimolar Tris-HCl at pH 7.5 by pipetting up and down to obtain the purified 3C library.
QPCR performed on the one experimental and two control 3C samples helped generate the digestion efficiency data. The 3C sample had an approximately 88%digestion efficiency across the seven genomic loci tested. Take the chromosome confirmation capture or 3C sample and controls and perform QPCR.
Excise the product bands corresponding to the expected fragment size. To perform gel extraction of the PCR products, use a commercial kit or follow the homemade protocol. For the latter, construct a homemade purification cartridge by poking a hole in the bottom of a 0.5 milliliter tube with a needle.
Pack a small amount of cotton into the bottom of the tube with the hole, filling no more than half of the tube. Place the tube into a 1.5 milliliter tube, ensuring that the smaller tube rests on the lip of the bigger tube and not in the tube. Carefully cut the gel fragment into smaller pieces and place it in the small tube of the cartridge.
Place the assembly in a minus 20 degrees Celsius freezer for five minutes. Then spin the assembly for three minutes at 13, 000 G and room temperature. Discard the small tube containing the agarose debris and keep the 1.5 milliliter tube with the extracted DNA in the buffer.
The samples were tested for the presence of long range chromatin contacts between the different genomic loci using combinations of loci-specific primers and QPCR. The product abundances relative to a control primer set were calculated and graphed for comparison. The data indicated that eight of the 10 reactions were conditional positives.
Gel purification and sequencing of the expected PCR product for the eight conditional positives and one negative reaction were gel purified and sequenced. The results for representative positive and negative reactions are shown.
