The overall goal of this procedure is to generate genome-wide maps of protein-DNA interactions in situ with high resolution and low background, without the need for formaldehyde crosslinking, chromatin solubilization, or antibodies. This method can help answer key questions in the transcription field, such as where various regulatory factors associate with chromatin, relative to gene-regulatory regions. The main advantage of this technique is that is provides high resolution genomic binding maps with negligible background, and avoids limitations associated with standard ChEC-seq protocols.
Demonstrating the procedure will be Sebastian Grunberg, my colleague from the Fred Hutchinson Cancer Research Center. In the afternoon or evening of the day prior to the experiment, start a pre-culture of the experimental use strain. Inoculate three milliliters of used peptone dextrose, or an appropriate selective medium with a single colony of the strain, bearing the MNase tagged factor.
Incubate this culture overnight in a 30 degrees Celsius shaker incubator. On the following morning, dilute the overnight culture into 50 milliliters of medium to an optical density at 600 nanometers of 0.2 to 0.3. Incubate the culture in the shaker incubator until it reaches an optical density at 600 nanometers of 0.5 to 0.7.
Prepare the needed amount of Buffer A plus additives, and keep it room temperature during the procedure. Prepare microfuge tubes for sample collection. For each new factor analyzed, collect samples without calcium addition, and add 30 seconds, one minute, 2.5 minutes, 5 minutes, and 10 minutes after calcium addition.
To each tube add 90 microliters of stop solution, and 10 microliters of 10%SDS. When the culture has reached an optical density at 600 nanometers of 0.5 to 0.7, decant the culture into a 50 milliliter conical tube, and centrifuge at 1, 500 times g for one minute at room temperature. Remove the supernatant, thoroughly resuspend the cells in one milliliter of Buffer A, and transfer the resuspended cells to a microfuge tube.
Pellet the resuspended cells in a microfuge at 1, 500 times g for 30 seconds at room temperature. Aspirate the supernatant, and thoroughly resuspend the cells in one milliliter of Buffer A by pipetting up and down. Pellet the resuspended cells at 1, 500 times g for 30 seconds at room temperature.
Remove the supernatant, resuspend the cells in one milliliter of Buffer A, and centrifuge again. After removing the supernatant, thoroughly resuspend the cells in 570 microliters of Buffer A.Add 30 microliters of 2%digitonin to facilitate permeabilization of the cells, and invert to mix. Permeabilize the cells in a heat block at 30 degrees Celsius for five minutes.
Pipette the reaction up and down to ensure an even distribution of cells. As a negative control, transfer 100 microliter aliquot of permeabilized cells to a microfuge tube containing stop solution and SDS, and vortex briefly to mix. Prior to beginning the time course it's critical to have the tubes of stop solution, the pipette, pipette-tips, and a timer next to the heat blocks so that the early time points can be collected efficiently.
To start the time course, add 1.1 microliters of one-molar calcium chloride to the permeabilized cells to activate MNase, and invert several times quickly to mix. Immediately return the mixed reaction to 30 degrees Celsius and start a timer. At each time point to be collected, pipette the reaction up and down to ensure an even distribution of cells, and then remove a 100 microliter aliquot of permeabilized cells to a microfuge tube containing stop solution and SDS.
Vortex briefly to mix. Once all time points have been collected, add four microliters of 20 milligrams per milliliter proteinase K to each sample. Vortex briefly to mix, and incubate at 55 degrees Celsius for 30 minutes.
Add 200 microliters of phenol:chloroform:isoamyl alcohol to each sample, vortex vigorously to mix, and centrifuge at maximum speed for five minutes at room temperature. Remove the aqueous phase to a new tube. Add 30 micrograms of glycogen, and 500 milliliters of 100%ethanol.
Vortex vigorously to mix, and precipitate on dry ice for 10 minutes, or until the solution is viscous. Pellet the precipitated DNA by centrifuging at maximum speed, and four degrees Celsius for 10 minutes. Decant the supernatant and wash each pellet with one milliliter of room temperature 70%ethanol.
Decant the ethanol and remove the remainder by inverting, and gently tapping the tubes on a paper towel. Briefly spin the samples using a bench-top centrifuge, then use a pipette to remove residual ehtanol, taking care not to disturb the pellet. Air-dry the pellets at room temperature for five minutes.
While the DNA pellets are drying, make a master mix of Tris buffer and RNase A for resuspending the pellets once they are dried, and vortex to mix. Add 30 microliters of the RNase A solution to each dried DNA pellet, vortex to mix, and incubate at 37 degrees Celsius for 20 minutes to digest RNa. After 20 minutes mix one microliter of 6X DNA loading dye with five microliters of each sample, and run on a 1.5%agarose gel at 120 volts for 40 minutes.
Begin the size selection procedure by adding to each sample, 75 microliters of SPRI beads in a polyethylene glycol solution. Pipette up and down 10 times to mix the beads and DNA mixture, and then incubate at room temperature for five minutes. During the SPRI bead incubation, prepare microfuge tubes containing 96 microliters of 10 millimolar Tris pH 8.0, and four microliters of five molar sodium chloride for each sample.
Place the tubes containing the beads and DNA mixture in a magnetic rack for two minutes to collect beads on the side of the tube, then transfer each supernatant to a new tube containing Tris and sodium chloride. Add 200 microliters of phenol:chloroform:isoamyl alcohol to each sample, vortex to mix, and centrifuge at maximum speed at room temperature for five minutes. Remove each aqueous phase to a new tube, add 30 micrograms of glycogen, and 500 microliters of 100%ethanol.
Precipitate DNA on dry-ice for 10 minutes, or until the solution is viscous. Pellet the precipitated DNA by centrifuging at maximum speed and four degrees Celsius for 10 minutes. Decant the supernatant and wash each pellet with one milliliter of 70%ethanol.
Decant the ethanol, removing the remainder by inverting and gently tapping the tubes on a paper towel. Briefly spin the samples using a bench-top centrifuge, and use a pipette to remove residual ethanol, taking care not to disturb the pellet. Air-dry the pellets at room temperature for five minutes.
Resuspend each dried pellet in 25 microliters of Tris pH 8. Determine the concentration of each sample using a high-sensitivity assay. Agarose gel electrophoresis of DNA from a chromatin endogenous cleavage analysis of Reb1, a general regulatory factor that binds nucleosome-depleted regions, reveals a calcium-dependent increase in DNA fragmentation over time as indicated by smearing, and eventual complete digestion of genomic DNA.
Visualization of fragment ends from a Reb1 chromatin endogenous cleavage sequencing experiment reveals robust enrichment of Reb1 over background, derived from the free MNase strain after 30 seconds of calcium treatment. Similarly, substantial DNA cleavage was observed by a fusion of the mediator subunit Med8 with MNase, but not free MNase-driven by the Med8 promoter one minute after calcium addition. To compare Reb1 chromatin endogenous cleavage sequencing data to chromatin immunoprecipitation and high throughput sequencing data, 1, 992 Reb1 peaks determined by the organic method were motif centered, and the average fragment end count at each base position in a 100 base pair window around the motif midpoint was determined.
A striking asymmetry and cleavage was observed with the majority of Reb1-MNase released fragment ends mapping to the upstream side of the motif. Once mastered, this technique can be done in one full working day, if it performed properly. When attempting this procedure it's important to remember to limit the number of samples assayed in parallel, to ensure accurate pipetting and handling times.
After its development, this technique paved the way for researchers in the field of transcription to, for the first time, unambiguously characterize the genomic association of a non-DNA binding, megadalton-size protein complex in yeast. After watching this video, you should have a good idea of how to perform the ChEC-Seq method for genome-wide mapping of protein-binding sites in the budding yeast genome.
