In-Nucleus Hi-C in Drosophila Cells

The nucleus Hi-C protocols allow the exploration of chromosomal confirmation at different resolution for the characterization of chromatin loops, domains, and compartment organizing the genome. This protocol provides high-resolution profiling of genomic interactions at different scales, yielding a more consistent coverage over the full range of genomic distances and data with less technical noise. Combining this protocol with genetic editing is a powerful strategy for testing the structural function of genetic elements.

It can also be applied to the characterization of structural variations that lead to disease. The nucleus Hi-C protocol can be applied to explore the genome organization of any eukaryotic genome. Doing SDS and Triton treatment disaggregates any nuclear clumps by pipetting as aggregates interfere with enzymatic reaction efficiency, and be sure to perform the appropriate quality controls measure prior to sequencing.

Begin by adding methanol-free formaldehyde to one times 10 to the seven Schneider's Line 2 Plus Drosophila cells in 17.5 milliliters of Schneider medium supplemented with 10%FBS to a final concentration of 2%Incubate the cells for 10 minutes at room temperature with mixing every 60 seconds or on a rotating wheel, and add glycine to a final concentration of 0.125 molar with mixing. After five minutes at room temperature and 15 minutes on ice, collect the cells by centrifugation and carefully resuspend the pellet in 25 milliliters of cold PBS and collect the cells with another centrifugation. At the end of the centrifugation, resuspend the cells in one milliliter of ice cold lysis buffer for counting and adjust the cells to a one times 10 to the six cells per milliliter concentration.

Incubate the cells on ice for 30 minutes, inverting the tube every two minutes to mix and collect the cells with another centrifugation. Resuspend the pellet in one milliliter of fresh ice cold lysis buffer. After centrifugation, wash the lysate with one milliliter of 1.25X restriction buffer.

Centrifuge again to collect the lysate. Resuspend the pellet in 360 microliters of fresh restriction buffer and 11 microliters of 10%SDS with careful pipetting and incubate for 45 minutes at 37 degrees Celsius and 7 to 950 revolutions per minute with occasional pipetting. At the end of the incubation, stop the permeabilization with 75 microliters of non-ionic surfactant and return the tube to the incubator for an additional 45 minutes with shaking at 950 revolutions per minute with occasional pipetting.

For chromatin digestion, add 200 units of Mbo1 to the tube for a four to 16-hour incubation at 37 degrees Celsius with rotation. At the end of the incubation, inactivate the enzyme with 20-minute incubation at 60 degrees Celsius. To fill in the restriction fragment overhangs and to label the DNA ends with biotin, add 1.5 microliters each of 10 millimolar dCTP, dGTP, dTTP, 20 microliters of 0.4 millimolar biotin dATP, 17.5 microliters of tris low EDTA buffer, and 10 microliters of five units per microliter of DNA polymerase one large fragment to the tube with careful mixing.

Incubate the reaction for 75 minutes at 37 degrees Celsius with shaking at 700 revolutions per minute every 10 seconds for 30 seconds. At the end of the incubation, add the ligation mix to the chromatin and adjust it to one milliliter final reaction volume with thorough gentle mixing and incubate overnight at 16 degrees Celsius. To degrade the sample proteins, add 50 microliters of 10 milligram per milliliter proteinase K to the tube for a two-hour incubation at 37 degrees Celsius.

At the end of the incubation, increase the temperature to 65 degrees Celsius overnight to reverse crosslink the sample. The next morning, degrade the RNA with 10 microliters of 10 milligram per milliliter RNAse A.After one hour at 37 degrees Celsius, add one volume of phenol chloroform to the tube and mix thoroughly by inversion to obtain a homogenous white phase. After precipitating the DNA according to standard protocols, quantify the DNA using a fluorogenic dye that binds selectively to DNA and a fluorometer according to the manufacturer's instructions.

To purify the DNA from undigested and digested aliquots, load 100 nanograms of undigested, digested, and ligated samples onto a 1.5%agarose gel and look for a smear centered around 500 base pairs in the digested sample versus a high molecular weight band for the ligated sample. To verify the Hi-C marking and ligation efficiency by amplification and digestion of a known interaction, after PCR, digest the products with Mbo1, Cla1, or both, and run the samples on a new 1.5 to 2%gel to allow estimation of the relative number of 3C and Hi-C ligation junctions. After sonicating the sample to obtain 200 to 500 base pair DNA fragments, transfer 130 microliters with five micrograms of DNA from each sample into new microcentrifuge tubes containing 16 microliters of 10X ligation buffer, two microliters of 10 millimolar dATP, five microliters of T4 DNA polymerase, and seven microliters of double distilled water for a 30-minute incubation at 20 degrees Celsius.

At the end of the incubation, add five microliters of 10 millimolar dNTPs, four microliters of 10X ligation buffer, five microliters of T4 polynucleotide kinase, one microliter of DNA polymerase one large fragment, and 25 microliters of double distilled water to the tubes for a second 30-minute incubation at 20 degrees Celsius. To select fragments mostly in the 250 to 550 base pair size range, perform sequential solid phase reversible and mobilization size selection with 0.6X, then 0.9X according to the manufacturer's instructions and elute the DNA with 100 microliters of tris low EDTA. For biotin pulldown for each library, wash 150 microliters of Streptavidin-linked magnetic beads two times with 400 microliters of 1X Tween buffer and rotate the samples for three minutes on a rotating wheel.

At the end of the incubation, resuspend the beads in 300 microliters of 2X no Tween buffer and mix with 300 microliters of Hi-C material for a 30-minute rotation on a rotating wheel. At the end of the incubation, wash the beads with 400 microliters of 0.5X Tween buffer for a three-minute incubation at 55 degrees Celsius and 750 revolutions per minute, followed by two washes in 200 microliters of 1X restriction buffer per wash. After the second wash, resuspend the beads in 100 microliters of dATP tailing mix for a 30-minute incubation at 37 degrees Celsius.

At the end of the incubation, resuspend the beads in 50 microliters of 1X ligation buffer and transfer the suspension to a new tube containing four microliters pre-annealed paired end adapters and two microliters of T4 ligase. After two hours at room temperature, remove the supernatant and wash the beads two times with 400 microliters of Tween buffer, then wash the beads one time with 200 microliters of no Tween buffer and one time with 100 microliters of restriction buffer before re-suspending the beads in 40 microliters of 1X restriction buffer. A smear of 200 to 1, 000 base pairs is observed when restriction with Mbo1 is successful.

If the ligation is successful, a high molecular weight band is observed at the top of the gel. Digestion efficiency can also be confirmed by quantitative PCR. An acceptable digestion efficiency is 80%or higher.

As illustrated, the amplification of a known medium range interaction in Hi-C ligation products can be estimated by digestion of the PCR product recovered in the amplification. A digestion efficiency of more than 70%is recommended to avoid having a large proportion of non-useful reads for the libraries after sequencing. The number of PCR cycles for the final amplification should be one cycle less than the number of cycles for which the smear is visible.

The level of digestion of the library indicates the abundance of valid Hi-C pairs and reflects the proportion of useful reads that will be obtained from the library. Using the unique valid Hi-C pairs, basic analysis of the pair distribution can be performed. As observed in this representative example, the NOTCH gene locus can be seen along with the architectural proteins, domains one and two, and histone modifications along the locus.

Upon deletion of the region containing both the CTCF and M1BP DNA binding sites, a dramatic change in chromatin contacts can be observed. After Hi-C experiment, a can be performed to reach a specific set of contacts, for example, from promoter regions. This is particularly useful when assessing large genomes.

As demonstrated, combining the Hi-C protocol with genetic addition can be used to assess the structural and regulatory function of genomic elements.