Centrifuge the FACS-isolated satellite cells and discard the supernatant. Wash the cells with one milliliter of PBS, centrifuge, and incubate them in one milliliter cold nuclear extraction buffer on ice for 20 minutes. Add 20 microliters of Concanavalin A coated magnetic beads into a 1.5 milliliter tube containing 850 microliters of cold binding buffer.
Then using a magnetic rack, wash the beads twice with one milliliter of cold binding buffer and allow the beads to accumulate at the side of the tube on the rack for five minutes before gently resuspending them in 300 microliters of cold binding buffer. Next, centrifuge the nuclei and resuspend them gently in 600 microliters of nuclear extraction buffer. Then mix 600 microliters of extracted nuclei with 300 microliters of Concanavalin A bead slurry and incubate at four degrees Celsius for 10 minutes.
After removing the supernatant using a magnetic rack, resuspend the bead bound nuclei with one milliliter of cold blocking buffer. Again, remove the supernatant and wash the bead bound nuclei twice with one milliliter of cold wash buffer. Separate the supernatant, gently resuspend the nucleus bead complexes with a specific primary antibody and incubate at four degrees Celsius overnight with gentle agitation.
The next day, remove the supernatant and wash the bead bound nuclei before resuspending in 100 microliters of cold wash buffer. Add 100 microliters of diluted protein A-micrococcal nuclease to the 100 microliters of bead bound nuclei and incubate at four degrees Celsius for one hour with agitation. After incubation, remove the supernatant and wash the bead bound nuclei twice before resuspending them in 150 microliters of cold wash buffer.
Next, add three microliters of 100 millimolar calcium chloride, mix quickly by flicking and incubate on ice for 30 minutes. Stop the reaction by adding 150 microliters of stop buffer and incubate at 37 degrees Celsius for 20 minutes. Centrifuge the samples and transfer the supernatant to a new microcentrifuge tube, discarding the pellet and beads.
Then add three microliters of 10%sodium dodecyl sulfate and 2.5 microliters of 20 milligrams per milliliter proteinase K.Mix by inversion and incubate for 10 minutes at 70 degrees Celsius. Then add 300 microliters of phenol:chloroform:isoamyl alcohol and vortex before transferring to two milliliter phase lock tubes. Centrifuge and add 300 microliters of chloroform to the same tube.
Centrifuge again and collect the supernatant into a 1.5 milliliter tube. Next, add one microliter of glycogen, then 750 microliters of 100%ethanol, and allow the DNA to precipitate overnight at minus 20 degrees Celsius. The next day, pellet the DNA by centrifugation, then wash the pellet with one milliliter of 100%ethanol and centrifuge twice before removing residual ethanol with a pipette.
Air dry the pellet for five minutes and resuspend it in 25 microliters of tris-EDTA buffer. The H3KME2 and H3K27AC were enriched around the TSS of PAC7, ITGA7, LAM2, CXCR4, and VCAM1. However, H3K4ME2 and H3K27AC were not enriched at those of ITGAM, PTPRC, PECAM, CKM, or MHY3.
Almost no signal was obtained with the IgG sample. HOMER known motif analysis AR peaks and satellite cells and percent targeted regions are shown. Seeker analysis unveiled almost 500 peaks with a peak score higher than 50 with more than 200 of them with a score higher than 100.
