Discovering CsgD Regulatory Targets in Salmonella Biofilm Using Chromatin Immunoprecipitation and High-Throughput Sequencing (ChIP-seq)

Chromatin immunoprecipitation followed by sequencing, or ChIP-seq, is a technique that can be used to discover the regulatory targets of transcription factors, histone modifications, and other DNA-associated proteins. In ChIP-seq, cells are harvested and DNA-binding protein. is crossed-linked to DNA in vivo with formaldehyde.

Cells are lysed, releasing the cell contents and chromatin is sheared into fragments of less than 1000 base pairs, usually between 200 and 600 base pairs. DNA, interacting with the target protein, is immunoprecipitated using antibody and isolated by de-crosslinking. DNA protein crosslinks are reversed and RNA and protein are digested.

The DNA is purified, made into libraries, and sequenced. ChIP-seq data can also be used to find differential binding of transcription factors in different environmental conditions or cell types. Initially, ChIP was performed through hybridization on a microarray.

However, ChIP-sequencing has become the preferred method due to technological advancements, decreasing financial barriers to sequencing, and massive high-quality data output. In this protocol, we will demonstrate techniques of performing ChIP-seq with bacterial biofilms, a major source of persistent and chronic infections. ChIP-seq will be performed on salmonella typhimurium biofilm and planktonic cells, targeting the master biofilm regulator, CSGD, to determine differential binding in the two cell types.

In this video, we will demonstrate techniques of determining the appropriate amount of biofilm to harvest, normalizing to a planktonic control sample, homogenizing biofilm for crosslinker access, and performing routine ChIP-seq steps to obtain high-quality sequencing results. Streak plate salmonella enterica serve our typhimurium on an LB agar plate to isolate colonies and incubate at 37 degrees Celsius overnight. Inoculate five mL LB broth with one to three colonies from the streak plate and incubate at 37 degrees Celsius with shaking for seven hours or to log growth.

Find the optical density of the flask culture at 600 nanometers, using a spectrophotometer and add one OD 600 equivalent of cell culture or 10 to the nine cells to an Erlenmeyer flask containing 100 milliliters of one percent tryptone. Incubate at 28 degrees Celsius with shaking for 13 hours. Biofilm cells appear as aggregated flakes and single planktonic cells are in the cloudy media.

Biofilm are highly resistant and stick to the sides of tubes and pipette tips. We use conditioned media to move biofilm initially and use warm PBS immediately before crosslinking to remove protein crosslinking substrates in the media. Researchers may need to find an appropriate solution to re-suspend biofilm in.

Collect the flask culture in a centrifuge tube and centrifuge at 12, 000 G for 10 minutes at 10 degrees Celsius. Decant the supernatant into a 0.2 micrometer filter unit and vacuum filter. Aliquot flask culture into tubes and centrifuge at slow speed to separate the two cell types.

Biofilm cells are in the pellet at the bottom of the tube and single cells are in the cloudy supernatant. Pipette the supernatant containing planktonic cells into a centrifuge tube for later. 25 micrograms of DNA is recommended as input for ChIP-seq experiments.

In our case, 30 milligrams of biofilm yield approximately 25 micrograms of DNA. Since biofilm aggregates have an abundance of proteinaceous extracellular material, it competes for crosslinker and may result in unequal crosslinked product presented for immunoprecipitation. A protein assay to determine equivalent cell material by protein concentration is suggested.

In this case, six OD 600 planktonic cells are harvested as a control for 30 milligrams of biofilm. Biofilm is harvested by wet weight and the colony farming units of biofilm can be found using the conversion factor shown. Researchers are encouraged to find the conversion factor of the biofilm forming species they work with by homogenization and drop dilutions.

Re-suspend the biofilm pellet in one milliliter of conditioned tryptone and move into a pre-weighed two milliliter snap cap or a screw cap tube. Centrifuge for one minute at 11, 000 G.Remove the supernatant from the tube and weigh the tube accurately. Subtract the tube weight from the weight of the tube with biofilm to find the weight of aggregates.

It should be within 10%of the target biofilm weight. Add one milliliter of PBS and vortex to re-suspend biofilm aggregates. Dispense the supernatant from slow speed centrifugation into centrifuge tubes.

Measure the optical density of the planktonic cells at 600 nanometers using a spectrophotometer and calculate the required volume for a final OD 600 of six. Cool the Flora centrifuge to 10 degrees Celsius and pellet the planktonic cells using the Flora centrifuge. Centrifuge at 10, 000 G for 10 minutes at 10 degrees Celsius.

Remove the supernatant and re-suspend the pellet in PBS. Remeasure the OD 600 of the planktonic cells using a spectrophotometer and dispense the volume of six OD 600 planktonic cells into a two milliliter snap cap or screw cap tube. Biofilm aggregates must be broken apart to allow crosslinker to access cells.

Planktonic cells are treated the same way as biofilm cells to reduce variables. Using metal beads in a mixer mill may be more effective for breaking apart biofilm than glass tissue homogenizers. Aseptically, add one sterilized metal bead to each of the tubes.

Homogenize using a mixer mill for five minutes at 30 Hertz. Observe aggregate tubes to confirm that the biofilm has been broken apart before transferring the homogenized cells to a new 1.5 milliliter tube, avoiding the metal bead. Bring the volume to one milliliter with PBS.

At this point, it is optional to perform drop dilutions to enumerate input cells. Dispense fresh formaldehyde into sample tubes to a final concentration of one percent. Incubate for 30 minutes at room temperature on a rotating wheel.

Add glycine to a final concentration of 125 millimolar to quench cross-linking and incubate for five minutes at room temperature on a rotating wheel. To wash the cells and remove excess crosslinker, centrifuge for three minutes at 8, 000 G and remove the supernatant. Re-suspend the pellet in protease inhibitors and 500 microliters of filter sterilized PBS.

Re-suspend the pellet in 600 microliters of lysis buffer and incubate on ice for 10 minutes. Move the partial lysate to a new tube containing 1.4 milliliters of IP dilution buffer. Keep on ice for one and a half to two hours and vortex occasionally.

Some resistant material may remain in the tubes. However, a long incubation period and occasional vortexing will break apart aggregated material and the remainder will be broken during sonication. Tune the sonicator.

Place the 15 milliliter tube in a beaker of ice and place the probe inside the tube. Pulse at 20 to 40 percent for five sonication bursts of 30 seconds and cool between sonication rounds. The cell lysate will appear cloudy before sonication and will appear clear after sonication.

To remove precipitated material, centrifuge at 8, 000 G for 10 minutes at four degrees and dispense the supernatant into a new tube, avoiding the black pellet. Sonicator energy transfer and cell type resistance may differ so a sonication assay is recommended to find the number of sonication rounds that will produce the fragment size range required for downstream library preparation and sequencing. Dispense sonicated DNA into one 1.35 milliliter aliquot for immunoprecipitation and one 200 microliter aliquot as input DNA.

Keep the input control at minus 80 degrees Celsius until de-crosslinking and digesting steps. After testing the antibodies to ensure their specificity for the target protein, add the antibody to immunoprecipitation tubes and incubate at four degrees Celsius overnight on a rotating wheel. Add 50 microliters of protein G magnetic beads and incubate at four degrees Celsius on a rotating wheel for three hours.

Bind the beads to the sides of tubes using a magnetic stand and perform washes. Wash twice with 750 microliters of cold IP wash buffer one. Wash one with 750 microliters of cold IP wash buffer two.

And wash twice with 750 microliters of cold TE at pH eight. Keep the tubes on the magnetic stand during the washes. Add 450 microliters of IP elution buffer to each tube and incubate at 65 degrees Celsius for 30 minutes with gentle vortexing every five minutes.

Bind the beads to the sides of tubes using a magnetic stand. Wait at least two minutes until the solution is clear and then dispense the cleared solution to a new 1.5 milliliter tube. To reverse crosslinks and digest RNA, add two micrograms of RNase eight and add sodium chloride to your final concentration of 0.3 molar in each tube.

Incubate at 65 degrees Celsius for more than six hours or overnight. To digest protein, add 180 micrograms of proteinase K to each tube and incubate at 45 degrees Celsius for three to five hours. Purify the DNA.

Magnetic beads are preferred for isolating the small amounts of DNA recovered during ChIP with bacterial cells. However, column based or phenylchloroform extraction may adequately recover ChIP DNA. After purification, libraries are prepared from purified ChIP DNA using a kit that is compatible with the selected sequencing platform.

ChIP DNA libraries often require adding the minimum amount of adaptors and performing the maximum number of PCR amplification cycles. Check library size by a bioanalyzer and check library concentration with a cubit or a QPCR library quantification kit. Pool the libraries and proceed with sequencing according to the selected platform’s specification.

Analysis of ChIP-seq data requires scoring base quality, trimming low-quality bases, aligning an assembly reads to a reference genome, calling peaks, associating peaks with genes, and analyzing over-represented binding motifs. ChIP-seq data usually has a low level of background. DNA that is controlled by the protein targeted by immunoprecipitation will be enriched and will appear as peaks that are at least two-fold above background.

In this video, we have described methods to perform ChIP-seq on salmonella biofilm and single planktonic cells which yields peaks of enriched DNA at genes controlled by the transcription factor of interest. The majority of bacterial life in nature is thought to exist in biofilms and up to 40%of human diseases are thought to be biofilm related. So they have enormous medical and economic impacts.

However, biofilm is resistant and more difficult to enumerate, break open, and to manipulate. Researchers can use the ChIP-seq techniques we described to harvest an appropriate amount of biofilm, homogenize the biofilm, lyse cells, sonicate, perform immunoprecipitation, purify and sequence DNA from other biofilm-forming bacterial species.