The overall goal of this chromatin immunoprecipation protocol is to determine the abundance and localization of histone modifications at defined regions throughout the budding yeast genome. This method can help answer key questions in chromatin regulation, such as how histone modifications are linked to gene expression control and how these modifications change in response to different environmental conditions. An extremely versatile protocol that can be used to investigate many different histone modifications and multiple E strains at one time.
Though this method can provide insight into histone modifications, it can also be applied to investigating other chromatin bonding proteins through the use of epitope tags or available antibodies. Start this procedure by growing the yeast cells inoculate 10 milliliters of YPD medium with a single colony of each appropriate strain, and grow it 30 degrees celsius in a shaking incubator at 220 rpm overnight. On the following day after measuring the optical density at 600 nanometers or OD600 of the culture, dilute the culture to an OD600 of 0.2 in 100 mL of YPD in a new flask.
Grow the cells at 30 degree celsius in a shaking incubator at 220 rpm until they reach mid log phase. When the cultures have reached mid log phase, add 2.7 milliliters of 37%formaldehyde directly to the medium in each flask for a final concentration of 1%formaldehyde. Transfer the flasks to a shaker at room temperature, and shake at 50 rpm for 15 minutes.
Add five milliliters of 2.5 molar glycine to the medium and continue shaking at 50 rpm at room temperature for five minutes to quench the formaldehyde. Subsequently, spin down the cells and wash them as described in the text protocol. Remove the supernatant after the last spin and proceed to making yeast lysates.
Re-suspend the cells in one milliliter of cold chromatin amino precipitation or ChIP Lysis buffer with 1 millimolar PMSF and 1:1000 dilution of a yeast protease inhibitor cocktail. Transfer the suspension to a 2.0 milliliter screw cap tube containing 200 microliters of glass beads. Transfer the cells to a bead-beating apparatus for a high speed agitation at four degree celsius.
Bead-beat for 30 seconds and ice for one minute. Bead-beat a total of six times, keeping the cells on ice for at least one minute between each 30 second beat-beating. Using a gel loading tip, transfer the lysate to a 1.5 milliliter tube.
Centrifuge the lysate at 15, 500 g at four degree celsius for 20 minutes. Discard the supernatant and re-suspend the pellet in 250 microliters of micrococcal nuclease digestion buffer by gently pipetting up and down. Add 2.5 microliters of micrococcal nuclease to each reaction.
Mix gently by inverting four to six times and immediately incubate in a 37 degree celsius water bath for 20 minutes. After 20 minutes, stop the reaction by placing the tubes on ice, adding five milliliters of 0.5 molar EDTA for a final concentration of 10 millimolar EDTA and mixing gently by inversion. Centrifuge at 15, 500 g at four degree celsius for 15 minutes.
Then, transfer each supernatant to a new 1.5 milliliter tube. For the most consistent results, it is critical to ensure that the same concentration of protein is used in each amino precipitation. Depending on the number of amino precipitation or IP reactions, make a master mix of each micrococcal nuclease released chromatin fraction and ChIP lysis buffer and allocate one milliliter containing 50 micrograms of total protein to individual tubes.
Remove 100 microliters from each IP mix to process as the input sample. Freeze the input samples at 20 degree celsius for later use. It is also important to make sure the same amount of beads bound to the antibody is used for each immunoprecipitation.
Using a wide board pipette tip, add 20 microliters of magnetic protein AG beads, pre-bound with antibody to each IP sample. Rotate the samples at eight rpm at four degree celsius for three hours. When the IP is done, place the tubes in a magnetic stand and let beads collect on the side of the tube.
Use a pipette to remove the supernatant. Add one milliliter of ChIP lysis buffer to the beads and rotate at eight rpm at four degree celsius for five minutes. Then, place the tubes on the magnetic stand and remove the supernatant.
In this manner, wash the beads successively with the following buffers for the number of times indicated. ChIP Lysis Buffer, High Salt Wash Buffer, LiCl/detergent Wash Buffer and TE.With the last TE wash, use 0.5 milliliters of TE to transfer most of the beads to a new tube, and then use another 0.5 milliliters of TE to wash the tube and transfer the remaining beads. Add 250 microliters of freshly made ChIP elution buffer to each tube and vortex the solution briefly.
Rotate the samples at eight rpm at room temperature for 15 minutes. Place the tubes in the magnetic stand. Carefully transfer the supernatant to a new tube and avoid the beads.
Add another 250 microliters of ChIP elution buffer to the beads. Rotate the samples at eight rpm at room temperature for 15 minutes. Carefully and without disturbing the beads, transfer the supernatant to the tube containing the first elution.
Thaw the input samples collected earlier and add 400 microliters of ChIP elution buffer to each input sample. To reverse protein DNA cross links, add to both input and IP samples, 20 microliters of five molar sodium chloride, five microliters of glycogen and 12.5 microliters of Proteinase K.Mix well by inverting or flicking the tubes. Incubate the samples in a 65 degree celsius water bath overnight.
To begin this procedure, make a QPCR master mix for each set of primers by combining the appropriate volumes of QPCR mix, the forward primer, and the reverse primer. Make DNA master mixes for each DNA sample where one reaction contains 0.5 microliters of DNA and 4.0 microliters of nuclease free water Add 5.5 microliters of the appropriate primer master mix to each well in a 384 well QPCR plate. Spin the plate at 200 g at room temperature for one minute.
Then, add 4.5 microliters of the appropriate DNA master mix to each well. Seal the plate, and spin at 200 g at room temperature for one minute. Perform QPCR using a real time QPCR system with the following conditions, 95 degree celsius for three minutes 40 cycles of 95 degree celsius for 10 seconds and 55 degree celsius for 30 seconds and a melting curve of 65 degree celsius to 95 degree celsius with 0.5 degree celsius increments for five seconds.
One key component of this protocol is optimizing the concentration of micrococcal nuclease used to digest the chromatin into soluble fragments. This example result from agarose gel electrophoresis of DNA isolated following digestion of the chromatin pellet with varying amounts of micrococcal nuclease shows that 2.5 microliters of enzyme at 20 units per microliter produced predominantly mononucleosomes. This representative ChIP experiment used antibodies against a histone methyl marker H3K4me2 and histone H3 in wild type and set1 knockout cells.
Set1 knockout cells lack the methyl transferase that catalyzes the mark The bars indicate the relative enrichment of H3K4me2 at the indicated low sci. The wild type strain showed clear enrichment for H3K4me2 at the five prime end of two genes known to be direct targets of set1, PMA1 and ERG11 whereas no signal was observed in set1 knockout cells. As expected, there was no enrichment of the H3K4me2 mark at TEL07L, the expression of SPR3 has also been reported to be regulated by set1 but these results indicate the regulation is likely to occur by a different mechanism than at PMA1 or ERG11.
Once mastered, this technique can be done within four days if it is performed properly. While attempting this procedure, it's important to remember to carefully test the antibody used for specificity with the proper controls. If available, both positive and negative control regions should be probed in the QPCR.
Following this procedure, other methods like GP sequencing can also be performed in order to determine the genome by distribution of histone modifications. After watching this video, you should have a good understanding of how to consistently detect the levels of different histone modifications at unique genomic regions with well controlled experiments.
