Encyclopedia of Experiments: Biological Techniques
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After counting in a hemocytometer, add 20,000 purified OPCs in 1 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 5 minutes at room temperature. Adding the glycine will stop the DNA-protein crosslinking. Then, wash the cross-linked cells with 1 milliliter of HBSS buffer with protease inhibitor cocktail. Next, centrifuge the cells in a pre-cooled centrifuge machine at 300 times g at 4 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 4 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 5 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 4 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 1 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 4 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 4 degrees Celsius for another 2 hours on the rotating wheel.
After 2 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 4 minutes on a rotating wheel at 4 degrees Celsius. Then, add 200 microliters of elution buffer to the bead pellet. Incubate the reaction tube at 65 degrees Celsius for 4 hours.
Once the double-stranded DNA is denatured, dephosphorylate the 3-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.
