זיהוי אתרים פקטור שעתוק Olig2 מחייב גנומית בחריפות מטוהר PDGFRα + תאים על-ידי Immunoprecipitation נמוך-תא כרומטין רצף ניתוח

The overall goal of this experiment is to analyze the genome-wide binding of oligodendrocyte transcription factor two in acutely purified brain oligodendrocyte precursor cells by performing immunopanning, low-cell chromatin immunoprecipitation, high-throughput sequencing, and bioinformatic data analysis. This method is a powerful tool for studying genome-wide transcriptional regulation and epigenetic mechanisms, which play important roles in cell differentiation and development. The main advantage of this technique is that it is broadly applicable in ChIP-seq of other transcription factors with any cell type of limited numbers, including acutely purified primary cells without proliferation in vitro.

Demonstrating the procedure will be Yanan You, a postdoc from my laboratory. She is the co-author of this paper. To select the PDGFR-alpha-positive cells, prepare one plate for immunopanning.

Prepare another two plates for endothelial cells and microglia depletion. Then, coat a 10-centimeter Petri dish with 30 microliters of goat anti-rat immunoglobulin. Then, agitate the plate in order to ensure that the entire surface of the plate is evenly covered with the coating solution.

Leave the Petri dish overnight at four degrees Celsius. Next, dilute 40 microliters of rat anti-PDGFR-alpha antibody with 12 milliliters of DPBS containing 0.2%bovine serum albumin. Next, wash the immunoglobulin G-coated plate with 10 milliliters of 1X DPBS for three consecutive times.

Then, incubate the immunoglobulin G-coated plate with PDGFR-alpha antibody solution at room temperature for four hours. After four hours, carefully add DPBS along the side wall of the plate to wash the rat anti-PDGFR-alpha antibody-coated plate three times. Do not disturb the coated surface of the plate.

Next, use 20 milliliters of DPBS with 2.3 micrograms per milliliter of BSL-1 to coat two 15-centimeter Petri plates for two hours. After incubation, carefully add 20 milliliters of DPBS along the side wall of the plate to wash the BSL-1 plate three times. Do not disturb the coated surface.

After washing, centrifuge the single-cell suspension at 300 times g for 10 minutes at room temperature. Post-centrifugation, add 15 milliliters of immunopanning buffer to the cell pellet. Then, incubate the single-cell suspensions from two mouse brains sequentially on two BSL-1-coated Petri dishes.

Then, leave the plates for 15 minutes at room temperature. Agitate the plate every five minutes during the incubation. Then, gently swirl the plate to collect all the non-adherent cells from the cell suspension.

Then, seed the cells on the rat PDGFR-alpha antibody-coated plate. Incubate the plate for 45 minutes at room temperature. At the end of 45 minutes, swirl the plate again.

Then, discard the cell suspension. Next, rinse the plate with DPBS for eight times to remove the non-adherent cells. Examine the plate under a microscope.

Next, add cell detachment solution on the rat PDGFR-alpha antibody-coated plate. Then, incubate the plate at 37 degrees Celsius for 10 minutes. In the meanwhile, shake the plate to dislodge the adherent cells, and examine it under the microscope.

Next, centrifuge the cells at 300 times g at room temperature. After centrifugation, resuspend the cell pellet with one milliliter of OPC cell culture medium. Then, count the cells utilizing the trypan blue exclusion method in a hemocytometer.

After counting in a hemocytometer, add 20, 000 purified OPCs in one milliliter of OPC cell culture medium for the ChIP reaction. Then, add 27 microliters of 36.5%of formaldehyde at room temperature for 10 minutes. This will fix the cells.

After 10 minutes, add 50 microliters of 2.5 molar glycine on the fixed cells for five minutes at room temperature. Adding the glycine will stop the DNA-protein cross-linking. Then, wash the cross-linked cells with one milliliter of HBSS buffer with protease inhibitor cocktail.

Next, centrifuge the cells in a pre-cooled centrifuge machine at 300 times g at four degrees Celsius. After cross-linking the purified OPCs, the cross-linked cells should be washed using ice-cold HBSS solution, and the remaining ChIP steps should be carried out at four degree, otherwise the antibody cannot precipitate the genomic DNA properly. Then, add 25 microliters of complete lysis buffer to the cell pellet, and leave on ice for five minutes.

Next, pipette 75 microliters of ice-cold HBSS buffer with protease inhibitor cocktail. Then, shear the chromatin of the cell lysate in a pre-cooled sonication system with five cycles of 30 seconds on and 30 seconds off program. Once the chromatin is sheared, centrifuge the cell lysate at 14, 000 times g for 10 minutes at four degrees Celsius.

At the end of the centrifugation, collect the supernatant. Then, dilute 100 microliters of the sheared chromatin with an equal volume of the ice-cold ChIP buffer with protease inhibitor. Next, add one microliter of rabbit anti-Olig2 antibody to 180 microliters of the diluted sheared chromatin.

Incubate the tubes on a rotating wheel for 16 hours at four degrees Celsius at 40 revolutions per minute. Next, add 10 microliters of pre-washed protein A-coated beads to the ChIP reaction tube in a cold room. Again, incubate the ChIP tubes at four degrees Celsius for another two hours on the rotating wheel.

After two hours, leave the ChIP reaction tube on the magnetic rack for a minute. Then, wash the bead pellet with 100 microliters of four wash buffers each for four minutes on a rotating wheel at four degrees Celsius. Then, add 200 microliters of elution buffer to the bead pellet.

Incubate the reaction tube at 65 degrees Celsius for four hours. Once the double-stranded DNA is denatured, dephosphorylate the three-prime end of the single-strand DNA by adding shrimp alkaline phosphatase. Then, add poly-T tail to the single-stranded DNA using terminal deoxynucleotidyl transferase.

Next, anneal the DNA poly-dA primer to the single-stranded DNA template. Next, amplify the ChIP-sequence library using forward and reverse primers for indexing. The number of PCR cycles for library preparation depends on the amount of starting DNA.

A good library preparation requires accurate quantification of input DNA amount. Too many or too few PCR cycles can influence library concentration, as well as the complexity, leading to PCR artifacts. Use paramagnetic beads to select the fragments ranging from 250 to 500 base pairs from the PCR-amplified ChIP-sequence library.

Use a microfluidic chip-capillary electrophoresis device to examine the quality of the selected ChIP-sequence library. To evaluate the purity of the immunopanned OPCs, immunostaining studies are performed. On using the OPC lineage marker NG2 antibody, it shows that the majority of the PDGFR-alpha antibody immunopanned cells are NG2 positive.

Next, quantitative PCR is done to verify the enrichment of PDGFR-alpha. The plot obtained shows little expression of Tuj1, a neuronal marker indicating significant enrichment of PDGFR-alpha in comparison to the dissociated brain cells. Similar results are obtained showing little expression for all the other markers, such as GFAP, Mog, and Mbp, indicating enrichment of PDGFR-alpha.

Then, the quality of the ChIP-sequence library is analyzed. The electropherogram represents a clear peak for Olig2 transcription factor ranging from 250 to 500 base pairs after size selection of the library PCR product. Here, the histogram shows the tag clonality, to verify the quality control metrics obtained before peak calling.

The bar indicates that more than 90%of the reads are mapped to only one genomic location. Then, a strand correlation plot is obtained, which depicts an enriched peak corresponding to the predominant fragment length. In the plot, the red lines represent the read length at 50 base pairs and the fragment length at 130 base pairs, respectively, thereby indicating high-quality data.

Once mastered, this technique can be done in three days if it is performed properly. After its development, this technique paved the way for researchers to explore genome-wide DNA binding site of transcription factors and other proteins in primary cells or rare cells. After watching this video, you should have a good understanding of how to purify primary OPCs from mouse brain by immunopanning and how to perform ChIP-seq experiment by using low number of cells.