This method can help elucidate the molecular mechanisms underlying the regulation of candidate target genes via epigenetic modifications during fungal pathogenesis in plant pathology. Chromatin immunoprecipitation sequencing technology can efficiently detect DNA segments that interact with histones and transcription factors in the whole genome. Compared with other methods, it can improve efficiency and resolution.
Begin by adding 100 microliters of liquid complete medium to the oatmeal tomato Agger plates using a 100 microliter pipette. Using an inoculation loop, scrape the mycelia of the wild type strain and the knockout strain. Collect the mycelia debris and transfer them to 250 milliliters of liquid complete medium.
Wash the fungal hyphae with 500 milliliters of 0.7 molar sodium chloride solution. Then collect the fungal myceliUM and weigh it. Add approximately one milliliter of lysis enzyme permeation solution per one gram of the fungal mycelium.
Place the hyphae for licing at 28 degrees Celsius for three to four hours with shaking at 150 RPM. Wash the liced hyphae with 50 milliliters of 0.7 molar sodium chloride solution. Centrifuge the sample and discard the supernatant carefully.
Re-suspend the protoplast in 20 milliliters of 0.7 molar sodium chloride solution. Add 55 microliters of 37%formaldehyde drop by drop to two milliliters of sodium chloride buffer containing protoplasts for cross-linking until the final concentration is 1%To quench the unreacted formaldehyde, add 20 microliters of 10 times glycine to each tube. Discard the supernatant carefully after centrifugation.
Re-suspend the pellet in one milliliter of 0.7 molar sodium chloride solution. Discard the supernatant carefully after centrifugation and re-suspend the pellet in 750 microliters of RIPA buffer. Add 37.5 microliters of 20 times protease inhibitor.
Shear the chromatin by sonication for eight minutes using an ultrasonic homogenizer. After the sample has been ultrasonically broken, take out a part of the sample as input containing all DNA and protein released after the sample is sonicated. To analyze the length of the DNA fragments, run a 1%agarose gel electrophoresis after sonication.
After centrifugation, transfer the centrifuged supernatant to a 1.5 milliliter centrifuge tube and store it at negative 80 degrees Celsius for later use. Before performing the IP experiment, dilute each chromatin sample to a ratio of one to 10 with one times RIPA buffer. Pipette 50 microliters of super paramagnetic protein beads into a two milliliter centrifuge tube.
Place the tubes on a magnetic stand and let the magnetic beads precipitate. Then remove the supernatant. Add one milliliter of pre-cooled one times RIPA buffer to the tube and wash super paramagnetic protein beads three times.
Place the tubes on a magnetic stand and remove the supernatant. Add 100 microliters of one times RIPA buffer to each tube. Add 30 microliters of the chromatin sample, 100 microliters of super paramagnetic protein beads and four microliters of H3K4me3 antibody to the tube, mixing them well.
Place the samples on a rotary shaker to incubate them overnight at four degrees Celsius at 30 times G.Wash the super paramagnetic protein bead antibody and chromatin complex by resuspending the beads in one milliliter of one times RIPA buffer. Wash the sample with one milliliter of low salt immune complex wash buffer, then with one milliliter of high salt immune complex wash buffer. Rinse the beads with one milliliter of lithium chloride buffer and remove the supernatant.
Then add one milliliter of TE buffer to the tube. Place the tube again with one milliliter of TE buffer and remove the supernatant with a pipette. Add 100 microliters of elution buffer to each centrifuge tube.
Perform elution at 65 degrees Celsius for 15 minutes. Centrifuge for one minute at 10, 000 times G at four degrees Celsius and collect the supernatant into new centrifuge tubes. Add eight microliters of five molar sodium chloride to all the tubes and incubate at 65 degrees Celsius for four to five hours or overnight to reverse the DNA protein cross-links.
Add one microliter of Rnase A and incubate for 30 minutes at 37 degrees Celsius. Add four microliters of proteinase K to each tube and incubate at 45 degrees Celsius for one to two hours. Add 550 microliters of the fenal chloroform and isoamyl alcohol mixture to the centrifuge tube.
Centrifuge for 15 minutes at 10, 000 times G and aspirate the supernatant. Add 1/10 volume of three molar sodium acetate solution, 2.5 volumes of absolute ethanol and three microliters of glycogen to the tube. Place the sample in a refrigerator at negative 20 degrees Celsius overnight for precipitation.
On the next day, centrifuge the tubes and discard the supernatant. Wash the pellet three times with one milliliter of freshly prepared 75%ethanol at 10, 000 times G.Add 50 microliters of sterile deionized water to sufficiently dissolve the precipitate. Repair the DNA ends to generate blunt ended DNA using a DNA end repair kit, and instructions mentioned in the text manuscript.
To add adenine basis to the three prime ends, add 30 microliters of DNA, two microliters of water, five microliters of 10 times Taq buffer, 10 microliters of one millimolar dATP and three microliters of Taq DNA polymerase to a 0.2 microliter PCR centrifuge tube. Mix them and react in a PCR machine at 72 degrees Celsius for 10 minutes. Perform linker ligation by mixing 10 microliters of DNA, 9.9 microliters of water, 2.5 microliters of T4 DNA ligase buffer, 0.1 microliter of adapter oligo mix, and 2.5 microliters of T4 DNA ligase in a 0.2 microliter PCR centrifuge tube.
Incubate the tube at 16 degrees Celsius for four hours. To amplify the DNA using PCR primers, add 10.5 microliters of DNA, 12.5 microliters of two times high fidelity master mix. one microliter of PCR primer PE 1.0 and one microliter of PCR primer PE 2.0 to a 0.2 microliter PCR centrifuge tube and mix well.
The H3K4me3 signals of the mobre2, mospp1 and moswd2 deletion mutants were significantly decreased at its functional target regions. Compared to the wild type strain P131, the signals of enriched H3K4me3 chromatin immunoprecipitation sequencing reads in the mobre2, mospp1 and moswd2 deletion mutants were largely decreased. Following this procedure, chip on chip and chip qPCR can be performed.
They are generally used to screen the downstream target gene of a protein and study protein DNA interactions at known genome binding sites.
