The metered it's used for to perform efficient ChIP sequencing of both ester and non-ester proteins in brown lipid tissue isolated from mouse. The main advantage of the technique is that the protocol has been optimized for efficient immunoprecipitation of chromatin-associated complex from the lipid tissue. Before incision, spray the euthanized mouse with 70%ethanol.
Place the mouse with its back facing up, then make an incision along the neck. Next, located BAT directly under the skin between the shoulders and carefully remove the thin layer of white fat. Cross-link each BAT pad in 10 milliliters of PBS containing 1%formaldehyde for 15 minutes at 150 RPM at room temperature.
After 15 minutes, quench cross-linking by adding 2.5 molar glycine to the BAT, resulting in a final concentration of 125 millimolar. Place it on a shaker for five minutes, rotating at 150 RPM at room temperature. Subsequently, transfer the BAT pad to 500 microliters of hypotonic lysis buffer and add two stainless steel beads for each BAT pad.
Then, use a bead-based tissue homogenizer to shred the tissue. Start with five minutes at max power. If needed, extend the time until the tissue is homogenized and single cells are separated.
Transfer the sample into a new tube, and centrifuge at four degrees Celsius at 16, 000 G for 10 minutes. After 10 minutes, transfer the supernatant into a new tube and keep the cell pellet on ice. Centrifuge the supernatant at four degrees Celsius at 16, 000 G for 10 minutes to prevent loss of floating cells.
Then, discard the final supernatant and re-suspend both cell pellets together in 300 microliters of SDS lysis buffer with one times protease inhibitor. Incubate on ice for 10 minutes. Next, sonicate the lysates to generate DNA fragments 200 to 500 base pairs long.
After sonication, remove debris by centrifugation for 10 minutes at 16, 000 G at four degrees celsius. To perform immunoprecipitation, keep 1%of the chromatin solution aside as the ChIP input and keep the inputs overnight at four degrees Celsius. Next, add the ChIP antibody to one milliliter of chromatin solution and perform parallel immunoprecipitation with corresponding pre-immune IgG or normal IgG of the same species.
Alternatively, save one milliliter of chromatin solution for a no antibody control. Incubate overnight at four degrees Celsius with rotation. To collect the immune complexes, add 50 microliters of protein A agarose 50%slurry to the immune complexes and incubate for one hour at four degrees Celsius with rotation at 150 RPM.
After an hour, pellet the beads by centrifugation for one minute at 1000 times G at four degrees Celsius. Perform sequential washes of the beads on 0.45 micrometer PVDF centrifugal filter columns with buffers of increasing stringency by first transferring the beads in the columns with 500 microliters of low salt wash buffer. Then, pellet the beads in the columns for three minutes at 2500 G.Discard the flow-through.
Repeat the wash with high salt wash buffer according to the low salt wash procedures. Then, repeat the procedures with lithium chloride and finally with one times TE.After the washes are completed, elute the immune complexes by adding 300 microliters of elution buffer to the pelleted beads in the columns. Incubate them at room temperature for 30 minutes on a shaker at 400 RPM.
Subsequently, collect the elute by spinning down the columns for three minutes at 2500 G in a fresh tube. Reverse the cross-links by incubating the elute and inputs collected at 65 degrees Celsius overnight. To recover DNA, add 300 microliters of phenol chloroform IAA to the elute and inputs at a one-to-one ratio and vortex for 10 seconds.
Next, spin down at 21, 000 G at four degrees Celsius for 20 minutes. Keep the pellet and discard the supernatant. Wash the pellet with one milliliter of cold 70%ethanol.
Then spin down at 21, 000 G at four degrees Celsius for five minutes. Discard the supernatant and air dry the pellet. Re-suspend the pellet in 70 microliters of DNAse-free water for further procedures.
Here is an example of ChIP Q PCR using BAT isolated from wildtype or GPS2-A knockout mice. Since GPS2 has been found to be required for the expression of nuclear encoded mitochondrial genes in different cell lines, the recruitment of GPS2 was tested on two specific nuclear encoded mitochondrial genes. NDUFV1 and TOMM20 in BAT from mice as the examples of a tissue highly enriched in mitochondria.
GPS2 promoter occupancy was compared in GPS2-A knockout mice and wild type letter mates. An expected decrease was observed in GPS2 binding on selective target genes in the BAT from GPS2-A knockout mice. Using the protocol described here, an increased binding of RNA polymerase two and the repressive histone mark, H3K9ME3, was recorded in the BAT from GPS2-A knockout mice as compared to wild type letter mates.
These results confirm the recruitment of GPS2 on selected nuclear encoded mitochondrial genes and show that GPS2 is required for preventing the accumulation of H3K9ME3 and thus required for stalling of pole two transcriptional activity on target promoters. The protocol described here, even if specifically optimized for the brown adipose tissue, represents a valued tool for performing ChIP from other murine and human tissue. Don't forget that working with phenochloroform can be extremely hazardous.
Therefore, it's extremely important to always operate under the fume hood while using this reagent.
