Chromatin Immunoprecipitation: Difference between revisions

Line 71:

''Perform all steps in an ice bucket or in the cold room at 4°C.''

1. Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads:

==== Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads====

~~<nowiki> a.~~ Add 200 μl re-suspended magnetic bead slurry to a 1.5 ml microfuge tube on ice containing 1 ml cold

# Add 200 μl re-suspended magnetic bead slurry to a 1.5 ml microfuge tube on ice containing 1 ml cold PBS/BSA. Vortex briefly to mix well.

PBS/BSA. Vortex briefly to mix well.

# Place the microfuge tubes on the magnet rack and remove supernatants.

b. Place the microfuge tubes on the magnet rack and remove supernatants.

# Resuspend the beads in 1 ml cold PBS/BSA.

c. Resuspend the beads in 1 ml cold PBS/BSA.

# Repeat Steps b and c 3 times.

d. Repeat Steps b and c 3 times.

# Add 200 μl PBS/BSA to beads.

e. Add 200 μl PBS/BSA to beads.

# Add 5 μg primary antibody. Do not vortex beads after adding the antibody.

f. Add 5 μg primary antibody. Do not vortex beads after adding the antibody.

# Gently mix on a rotator platform for at least 2 hours at 4°C.

g. Gently mix on a rotator platform for at least 2 hours at 4°C.

# Wash beads 3 times (steps b-c), resuspending the beads by inverting the tubes during each wash.

h. Wash beads 3 times (steps b-c), resuspending the beads by inverting the tubes during each wash.

# Resuspend in 100 μl PBS/BSA, and proceed to Step 2.

i. Resuspend in 100 μl PBS/BSA, and proceed to Step 2.~~</nowiki>~~

2. Incubate bead-antibody complex with fragmented, cross-linked chromatin

==== Incubate bead-antibody complex with fragmented, cross-linked chromatin====

~~<nowiki> a.~~ Add 100 μl of antibody-coupled beads (from step 1.i above) to each 300 μl chromatin preparation (from

# Add 100 μl of antibody-coupled beads (from step 1.i above) to each 300 μl chromatin preparation (from Sonication protocol) and incubate on a rotator for one hour at room temperature, followed by one hour at 4°C.

Sonication protocol) and incubate on a rotator for one hour at room temperature, followed by one hour at

# Collect beads containing immuno-bound chromatin by placing the microfuge tube on a magnet rack.

4°C.

# Remove and discard supernatant.

b. Collect beads containing immuno-bound chromatin by placing the microfuge tube on a magnet rack.

# Wash beads 5 times with LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator.

c. Remove and discard supernatant.

# Add 1 ml TE Buffer to beads. Mix 1 minute on rotator and then place tubes on magnet rack to collect beads and discard supernatant.

d. Wash beads 5 times with LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator.

# Resuspend the bead pellet in 200 μl IP Elution Buffer (at room temperature). Vortex to mix.

e. Add 1 ml TE Buffer to beads. Mix 1 minute on rotator and then place tubes on magnet rack to collect beads

and discard supernatant.

f. Resuspend the bead pellet in 200 μl IP Elution Buffer (at room temperature). Vortex to mix.~~</nowiki>~~

3. Reverse cross-linking and recover ChIP DNA

==== Reverse cross-linking and recover ChIP DNA ====

~~<nowiki> a.~~ Incubate bead pellet from step 2.f above in a 65°C water bath for 1 hour, shake or vortex

# Incubate bead pellet from step 2.f above in a 65°C water bath for 1 hour, shake or vortex every 15 minutes to elute the immuno-bound chromatin from the beads.

every 15 minutes to elute the immuno-bound chromatin from the beads.

# Spin at 14,000 rpm in a microfuge at room temperature for 3 minutes.

b. Spin at 14,000 rpm in a microfuge at room temperature for 3 minutes.

# Collect the supernatant, which contains the ChIP’d DNA. The tubes can be placed on the magnet to facilitate supernatant recovery.

c. Collect the supernatant, which contains the ChIP’d DNA. The tubes can be placed on the

# Incubate the supernatant containing the ChIP’d DNA in a 65°C water bath overnight to complete the reversal of the formaldehyde cross-links.

magnet to facilitate supernatant recovery.

d. Incubate the supernatant containing the ChIP’d DNA in a 65°C water bath overnight to

complete the reversal of the formaldehyde cross-links.~~</nowiki>~~

===Analysis of Immunoprecipitated DNA===

@@ Line 71: / Line 71: @@
 ''Perform all steps in an ice bucket or in the cold room at 4°C.''
-. Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads:
+==== Couple the primary antibody for each transcription factor or chromatin protein to magnetic beads====
-   <nowiki> a. Add 200 μl re-suspended magnetic bead slurry to a 1.5 ml microfuge tube on ice containing 1 ml cold
+# Add 200 μl re-suspended magnetic bead slurry to a 1.5 ml microfuge tube on ice containing 1 ml cold PBS/BSA. Vortex briefly to mix well.
-       PBS/BSA. Vortex briefly to mix well.
+# Place the microfuge tubes on the magnet rack and remove supernatants.
-   b. Place the microfuge tubes on the magnet rack and remove supernatants.
+# Resuspend the beads in 1 ml cold PBS/BSA.
-   c. Resuspend the beads in 1 ml cold PBS/BSA.
+# Repeat Steps b and c 3 times.
-   d. Repeat Steps b and c 3 times.
+# Add 200 μl PBS/BSA to beads.
-   e. Add 200 μl PBS/BSA to beads.
+# Add 5 μg primary antibody. Do not vortex beads after adding the antibody.
-   f. Add 5 μg primary antibody. Do not vortex beads after adding the antibody.
+# Gently mix on a rotator platform for at least 2 hours at 4°C.
-   g. Gently mix on a rotator platform for at least 2 hours at 4°C.
+# Wash beads 3 times (steps b-c), resuspending the beads by inverting the tubes during each wash.
-   h. Wash beads 3 times (steps b-c), resuspending the beads by inverting the tubes during each wash.
+# Resuspend in 100 μl PBS/BSA, and proceed to Step 2.
-   i. Resuspend in 100 μl PBS/BSA, and proceed to Step 2.</nowiki>
-. Incubate bead-antibody complex with fragmented, cross-linked chromatin
+==== Incubate bead-antibody complex with fragmented, cross-linked chromatin====
-   <nowiki> a. Add 100 μl of antibody-coupled beads (from step 1.i above) to each 300 μl chromatin preparation (from
+# Add 100 μl of antibody-coupled beads (from step 1.i above) to each 300 μl chromatin preparation (from Sonication protocol) and incubate on a rotator for one hour at room temperature, followed by one hour at  4°C.
-      Sonication protocol) and incubate on a rotator for one hour at room temperature, followed by one hour at
+# Collect beads containing immuno-bound chromatin by placing the microfuge tube on a magnet rack.
-°C.
+# Remove and discard supernatant.
-   b. Collect beads containing immuno-bound chromatin by placing the microfuge tube on a magnet rack.
+# Wash beads 5 times with LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator.
-   c. Remove and discard supernatant.
+# Add 1 ml TE Buffer to beads. Mix 1 minute on rotator and then place tubes on magnet rack to collect beads and discard supernatant.
-   d. Wash beads 5 times with LiCl Wash Buffer, mixing 3 minutes for each wash on a rotator.
+# Resuspend the bead pellet in 200 μl IP Elution Buffer (at room temperature). Vortex to mix.
-   e. Add 1 ml TE Buffer to beads. Mix 1 minute on rotator and then place tubes on magnet rack to collect beads
-      and discard supernatant.
-   f. Resuspend the bead pellet in 200 μl IP Elution Buffer (at room temperature). Vortex to mix.</nowiki>
-. Reverse cross-linking and recover ChIP DNA
+==== Reverse cross-linking and recover ChIP DNA ====
-   <nowiki> a. Incubate bead pellet from step 2.f above in a 65°C water bath for 1 hour, shake or vortex
+# Incubate bead pellet from step 2.f above in a 65°C water bath for 1 hour, shake or vortex every 15 minutes to elute the immuno-bound chromatin from the beads.
-      every 15 minutes to elute the immuno-bound chromatin from the beads.
+# Spin at 14,000 rpm in a microfuge at room temperature for 3 minutes.
-   b. Spin at 14,000 rpm in a microfuge at room temperature for 3 minutes.
+# Collect the supernatant, which contains the ChIP’d DNA. The tubes can be placed on the magnet to facilitate supernatant recovery.
-   c. Collect the supernatant, which contains the ChIP’d DNA. The tubes can be placed on the
+# Incubate the supernatant containing the ChIP’d DNA in a 65°C water bath overnight to complete the reversal of the formaldehyde cross-links.
-      magnet to facilitate supernatant recovery.
-   d. Incubate the supernatant containing the ChIP’d DNA in a 65°C water bath overnight to
-      complete the reversal of the formaldehyde cross-links.</nowiki>
 ===Analysis of Immunoprecipitated DNA===