בידוד של אזורים הגנומי ספציפיים וזיהוי של Associated מולקולות על ידי enChIP

The overall goal of this procedure is to isolate a specific genomic region retaining molecular interactions for identification of molecules interacting with the genomic region. This method can help answer questions in the genomic functions fields such as transcription and epigenetic regulation. The main advantage of this technique is that it enables direct identification of molecules interacting with a specific genomic region.

Demonstrating the procedure will be Toshitsugu Fujita, an assistant professor from my laboratory. Following the text protocol, engineer the DNA-binding molecules of interest. This entails generating the gBlock sequence of interest and inserting it into a retrovirus for example to produce the guide RNA complexed with three FLAG-dCas9.

Alternatively, three FLAG-TAL protein recognizing the target sequence can be generated. Then, following the text protocol infect the cells with the prepared retrovirus. Confirm the expression of the engineered DNA-binding molecules and establish the cell line.

Now, proceed by cross-linking the cells with formaldehyde. Suspend 20 million transfected cells in 30 milliliters of regular culture medium and add 810 microliters of 37%formaldehyde for a final concentration of 1%Incubate the mixture for five minutes at 37 degrees Celsius. After five minutes, kill the cross-linking reaction by adding 3.1 milliliters of 1.25 molar glycine.

Let the cells sit at room temperature for 10 minutes before proceeding. Next, centrifuge the cell tube at 300 G for five minutes in a refrigerated centrifuge. Carefully discard the supernatant and wash the cell pellet in 30 milliliters of PBS by mixing and centrifugation.

Perform two PBS washes. If needed, the washed pellet of fixed cells can be stored at negative 80 degrees Celsius. Continuing on, collect the chromatin from the cells.

Suspend them in 10 milliliters of lysis buffer and incubate them on ice for 10 minutes. Then, using a refrigerated centrifuge, collect the cells and discard the supernatant carefully. Now, suspend the cell pellet in nuclear lysis buffer and incubate it on ice for 10 minutes.

Every few minutes during the incubation briefly vortex the mixture. Again, collect the cells by centrifugation. Now, wash the pellet with 10 milliliters of PBS and remove the wash solution with another centrifugation.

This produces the chromatin fraction and, if desired, can be flash frozen and stored at negative 80 degrees Celsius. Proceed by suspending the chromatin pellet in 800 microliters of modified lysis buffer three and transfer the suspension to a smaller 1.5 milliliter tube. Now, power up a sonication probe at level three power with continuous duty.

To fragment the chromatin, manually cycle between 10 seconds of sonication and 20 seconds on ice. On every 5th or 6th cycle, extend the ice phase to two minutes so the sample doesn't overheat. Perform this sonication cycle 10 to 18 times, keeping the tip of the probe off the bottom of the tube to avoid foaming up the sample.

After the sonication, collect the sample with a fast refrigerated centrifugation. Then, transfer the supernatant containing the fragmented chromatin to a new tube and discard the pellet. See the text protocol on how to evaluate the fragmentation.

To prepare for the immunoprecipitation, first conjugate the antibodies to Dynabeads. This immunoprecipitation is scaled to samples derived from 20 million cells. Add a 1/5 volume of modified lysis buffer three with Triton X-100 to the fragmented chromatin to get a final concentration of 1%Triton.

Next, add the mixture to the magnetic beads conjugated to normal mouse IgG. Incubate the reaction for an hour on a rotator with refrigeration. Later, attract the beads from the solution for three minutes on a magnet stand.

Then, collect the supernatant. Transfer the supernatant to the tube of magnetic beads conjugated to anti-FLAG antibodies. Run this conjugation reaction overnight on a rotator at four degrees Celsius.

The next morning, apply a magnet to the tube for three minutes to collect the beads. Then, pipette out the supernatant and discard it. Now, wash the beads.

Suspend them in one milliliter of low-salt buffer and incubate them for five minutes on a rotator at four degrees Celsius. Following the incubation, isolate the beads magnetization and discard the supernatant. Repeat this wash step with low-salt buffer once more.

Using the same technique, perform two more washes using high-salt buffer. Continue using the method to wash the beads in lithium chloride buffer twice. Finally, wash the beads with TBS-IGEPAL CA-630 just once.

After the seven wash steps, suspend the beads in 200 microliters of elution buffer and incubate them at 37 degrees Celsius for 20 minutes. Then, attract the beads by magnetization and collect the supernatant into a new tube. Repeat this elution step to increase the yields.

Then, purify about 5%of the DNA to estimate the yield. Begin by mixing the bulk of the eluate into a precipitation reaction. Precipitate the chromatin overnight at negative 20 degrees Celsius.

The next day, centrifuge the sample and discard the supernatant. Rinse the pellet with a milliliter of 70%ethanol and repeat the centrifugation for 10 minutes. Then, discard the supernatant and suspend the pellet in 40 microliters of 2x sample buffer.

Now, vortex the suspension for a full five minutes. Then, incubate it at 100 degrees Celsius for 30 minutes to denature and reverse the cross-linking. After the boil, run 40 microliters of the sample out on an SDS-PAGE gel.

Usually a four to 20%gradient is employed. Run the gel until the dye travels one centimeter below the well. Then, stain the gel and cut it into five pieces that are about 2 millimeters long.

Dilute the protein from the gel pieces and use it for mass spec analysis. Purification of the RNA, followed by sequencing analysis, are covered in the text protocol. Overall, up to 10%of target genomic regions can be purified using enChIP.

Seen here are yields of analyses targeting the promoter region of IRF-1 gene as compared to the non-specific Sox2 locus. Another example is the targeting telomeres versus non-specific gamma-satellite locus. enChIP SILAC identified several proteins associated with the IRF-1 promoter in an interferon gamma-specific manner.

Telomere-binding proteins were also identified by enChIP combined with mass spectrometry. enChIP RNA sequencing was then used to identify the RNAs associated with the telomeres. After its development, this technique paved the way for researchers in the fields of transcription and epigenetic regulation to explore such mechanisms in various biological systems.