Binding antibodies to magnatic beads
1， For each IP , use 10ul stock solution of beads, wash twice the protein A/G coated magnetic beads with ice cold ChIP Buffer. Prepare same amount of beads for each IP if pre-clearing is desired.
2， After washing, resuepend the beads in ChIP Buffer to the same concentration as stock.
3， Aliquot 90ul of ChIP Buffer per Ip to a mewly prepared tube
4， Add 10ul of pre-washed Protein A/G-beads per IP tube made up 100ul total per IP
5， Add specific antibody and control antibodies. The amount of antibody varies depends on the antibody used, the binding capacity of 10 ul magnetic beads is 3ug
6， Incubate the tubes at 40rpm on a rotating wheel for at least 2hours at 4℃.
Chromatin shearing and nuclear membrane solubilization
1， Seeding cells in 20ul Nuclei Extraction Buffer, invert tubes several times gently, then centrifuge for 5 minutes in 4000rpm in 4℃. Discard 10ul supernatant and keep another 10ul with nucleus.
2， Prepare MNase Master mix ；
3， Add 40ul MNase Master Mix to each sample ,mix well by pipetting or gentle vertex
4， Proceed at room temperature (25℃) for 10min
5， Add 5.5ul MNase Stop Solution,mix well by gentle vertex
6， Add 5.5ul Nuclear Break Buffer into the tube , mix well by gentle vertex. These are sheared chromatin ready to be ChIPed
7， Add Complete ChIP Buffer to make 100ul sheared chromatin for each IP and 10ul for input
Magnetic immunoprecipitation
1， Briefly spin the tube containing the antibody-coated beads to bring down liquid caught inside the lid.
2， Place tubes in the ice-cold magnetic rack , wait for 1min. discard the supernatant. Keep the antibody-coated beads
3， Add 100ul of diluted sheared chromatin to each IP tube, and keep 10ul of diluted chromatin as input at 4 ℃ or store at -20℃
4， Invert the tubes several times make sure beads are resuspended
5， Incubate samples at 4℃ under constant rotation on a rotator at 40 rpm for 2 hours up to overnight
Washes
1， Brief spin the tubes to bring down the liquid caught on the lid
2， Place the tubes into the magnetic rack, wait 1 min and discard the buffer
3， Wash twice using 100ul of ice-cold Low Salt Wash Buffer and twice using High Salt Wash Buffer
4， Add 100ul Hot Elution Buffer to the beads and 90ul Elution buffer to the input and transfer them to a new tubes
5， Put the strips in a PCR block at 65 for 1.5-2h
6， Place the strips on a magnetic rack, let sit for 1min, transfer them to a PhaseLock tube
DNA isolation
1， Brief spin the PhaseLock tube to bring down the agrose
2， Add 100ul DNA and equal volume(100ul) phenol:chloroform:isoamyl alcohol into the PhaseLock tube. Mix well by vertex
3， Spin at 13000g for 5min at room temperature
4， Transfer the supernatant to a new 1.5Ml tube
5， Add 1/10 of the vol.NaOAc and LPA
6， Add 2.5X Vol. ice-cold 100%EtOH. Mix well by vertex
7， Put the tubes at -20℃ to precipate for 30min up to overnight
8， Centrifuge DNA at 13000g for 30min at 4℃
9， Remove the supernatant and add 1ML 70% EtOH , let the tube sit for 5min to allow the salt to dissolve. Then centrifuge at 13000g for 5min at 4℃ to attach the pellet to the bottom.
10，Remove the EtOH and let the pellet to dry for 5min at room temperature
11， Add 30ul DNA elution Buffer