This protocol details all experimental procedures, from epitope tagging of transcription factor coding genes through computational data analysis, to perform genome-wide profiling of transcription factor-DNA binding interactions in Candida albicans via CUT&RUN. This is a more accessible, faster, more sensitive, less expensive technique, and provides higher quality data than traditional ChIP-chip and ChIP-seq approaches. Although our protocol is developed for Candida albicans cells isolated from biofilms or planktonic cultures, it should be practical to adapt it to virtually any good form of Candida albicans cells.
After incubating the cell suspension, transfer a five-microliter aliquot into a new PCR tube. To five microliters of isolated nuclei and a five-microliter aliquot of intact cells previously stored at four degree Celsius, add one microliter each of calcofluor-white and SYTO 13. Incubate at 30 degrees Celsius in the dark for 30 minutes.
Visually inspect the integrity and purity of the isolated nuclei using a fluorescence microscope. Look for isolated nuclei showing prominent SYTO 13 staining and ensure no cell wall staining by the calcofluor-white dye. Repeat the inspection with intact cells as a control for cell wall staining with calcofluor-white dye.
Place the tubes on a magnetic rack and wait until the slurry is completely clear. Discard the supernatant using a pipette. Add 50 microliters of the antibody buffer and gently mix by pipetting.
Add three microliters of the anti-GFP polyclonal antibody to the tubes and incubate the tubes on a nutating mixer at 4 degrees Celsius for two hours. Briefly centrifuge the tubes at 100 times g at room temperature for five seconds and place the tubes on a magnetic rack. Once the slurry is clear, discard the supernatant using a pipette.
While the tubes with the beads are still on the magnetic rack, add 200 microliters of ice-cold cell permeabilization buffer directly onto the beads. Discard the supernatant using a pipette. Repeat two washes with the ice-cold cell permeabilization buffer.
Add 50 microliters of ice-cold cell permeabilization buffer to each tube and gently mix by pipetting. Add 2.5 microliters of the protein AG-MNase to each sample and mix by pipetting. Place the samples on a nutating mixer at 4 degrees Celsius and incubate the samples for one hour.
Briefly centrifuge the strip tubes at 100 times g at room temperature for five seconds, then place the tubes on a magnetic rack. Once the slurry is clear, discard the supernatant using a pipette. While the tubes with the beads are still on the magnetic rack, add 200 microliters of ice-cold cell permeabilization buffer directly onto the beads.
Discard the supernatant using a pipette. Repeat two washes with the ice-cold cell permeabilization buffer. Add 100 microliters of the ice-cold cell permeabilization buffer to the samples and gently pipette up and down five times.
Remove the gel cast from the gel box and open the gel cast per the manufacturer's instructions. Gently remove the gel from the gel cast and place it inside a gel-holding tray containing 100 milliliters of single-strength TBE. Add 10 microliters of the nucleic acid gel stain to the tray and gently swirl.
Cover with foil to protect from light and incubate statically at room temperature for 10 minutes. Then rinse the gel twice with 100 milliliters of deionized tap water. Image the gel under blue light illumination using an amber filter cover.
For each library, cut the gel slightly above the approximately 125 base pair prominent adapter dimer band and below the 400 base pair ladder mark. Puncture the bottom of a 0.65-milliliter tube using a 22-gauge needle and place the punctured tube inside a sterile two-milliliter microfuge tube. Transfer the gel slice to the punctured tube inside the two-milliliter microfuge tube.
Centrifuge the two-milliliter microfuge tube containing the 0.65-milliliter punctured tube and sample at 10, 000 times g at room temperature for two minutes to collect the gel slurry inside the two-milliliter microfuge tube. Add 300 microliters of ice-cold gel elution buffer to the gel slurry. Mix on a nutator at room temperature for a minimum of three hours or overnight.
Transfer all liquid and gel slurry to a 0.22 micrometer filter column and centrifuge at 10, 000 times g at room temperature for one minute. Download the source code for the CUT&RUN analysis from the GitHub page by clicking on the green Code button, followed by Download ZIP option. Unzip the folder to a relevant location on the local machine.
Install Conda Environment and run only once. Once Conda is installed, create a virtual environment provided with the relevant command. Activate the virtual environment every time this workflow is executed.
Cell wall digestion and nuclear integrity were assessed by visualizing both control intact cells and isolated nuclei stained with fluorescent cell wall and nucleic acid stains. In contrast to the isolated intact nuclei where cell wall staining is not observed, both the nuclei and the cell walls are fluorescently labeled in the intact control cells. The figure shows CUT&RUN TF libraries analyzed using a capillary electrophoresis instrument.
Successful CUT&RUN TF libraries are enriched for short fragments smaller than 200 base pairs. Suboptimal CUT&RUN TF libraries show enrichment for large DNA fragments. The figure herein shows that Ndt80 DNA-binding motifs are enriched across all the Ndt80-bound loci identified by CUT&RUN, indicating that the additional peaks identified by this methodology are likely bonafide Ndt80-bound sites.
Comparative analysis indicated that the CUT&RUN protocol identified most of the previously known binding events for Ndt80 and Efg1 during biofilm formation. Overall, both Ndt80 and Efg1 bound to loci overlap with previously published chromatin immune precipitation ChIP data and are identified only using the CUT&RUN. It enables us to significantly increase the scope and pace of our research focused on transcriptional regulatory networks and Candida albicans.
