REVISED CHROMATIN IP PROTOCOL
In obscene detail with notes for
beginners
PRELIMINARY NOTES ABOUT GROWING UP CELLS:
(1)
For experiments with MyoD-ER, when you plate
cells for experiments, plate them in growth media
WITHOUT phenol red; it activates ER. Also, don’t
forget to add L-Glutamine- the DME w/o phenol red
lacks it.
(2)
For fibroblasts, grow til 80-90% confluent,
then ONE MORE DAY before inducing.
(3)
Two plates per condition is better than 1, but
count on 1 x 15cm dish per condition at least.
WHEN YOU’RE READY TO HARVESTCELLS: BY TRYPSINIZATION:
 Warm trypsin, DME+10% BCS (25 mL per
condition), PBS
 Prepare formaldehyde fix: Need 2.5 mL per
condition
For 5 mL, it should be 3.5 mL Fixation
buffer
1.5 mL 37%
Formaldehyde
(1)
Wash cells in PBS.
(2)
Trypsinize cells: 2 mL per 15cm dish. Keep
lids clean.
(3)
Quench trypsin with media with serum. If two
15cm plates, add 11.35 mL per plate (giving you a
grand total of 27mL including trypsin when you pool
the two plates). Pool the volume from the two
plates in a 50mL falcon tube.
(4)
Take out sample for RNA- take 1.7mL from 27mL
(should be 10-20% of total material). Leave this
in a microfuge tube on the side until the cells get
into fix.
(5)
Add fix (2.5mL to remaining 25mL media/cells),
mix by inversion, and INCUBATE on bench for 30’,
RT. If working with lots of samples, such that it
takes several minutes to process all the plates, do
one sample at a time from quench to fix, note the
time lag between samples, and later, quench the fix
at the same amount of time lag for the sample.
*While cells are crosslinking, spin out cells in
microfuge tube, decant sup, save pellets at –80ºC
for eventual RNA preparation.
(6)
Quench fix with 1.4mL of 2.5M Glycine per 25mL
media. Let sit for 3’. If staggered, put on ice
until last sample is done.
*NB: From this point on, try to minimize unnecessary
contact between cell mix/chromatin and the container,
as it sticks like no one’s business and you’ll only
lose your sample. Use low bind tips, if available,
and other such measures.
(7)
Spin in TC Room table top centrifuge at 1250
RPM for 5’.
(8)
Wash pellet in 40mL PBS, resuspending pellet by
gentle vortexing, spin at 1250 RPM, 5’. Aspirate
all but 0.5mL PBS with pellet.
(9)
Add 1mL of CELL WASH #2. Resuspend pellet and
transfer to 15mL conical tube. Wash off stickage
from 50mL tube with remaining 14 mL of cell WASH #2
buffer.
Let stand in wash for 10’. Pellet by
spinning 1800 RPM, 5’.
(10)
Add 2mL of CELL WASH #3.
Resuspend pellet by
vortexing, add remaining 13mL, invert to mix.
Incubate, 10’. Spin 1800RPM, 5’.
(11) Add 1mL of CELL WASH #3, resuspend, transfer to
microfuge tube, pellet, decant sup.
STOP HERE IF YOU WANT- FREEZE O/N, -80ºC.
Bennett thinks he gets better yields if he proceeds
to IP in one shot, but you may stop here, and you
won’t die from it.
(11b) Lyse pellet in 200L Dilution/Lysis buffer
containing protease inhibitors, and if you’re
looking at histone modifications, butyrate, too.
Resuspend well, put on ice.
(12)
Sonicate each sample: 6x 20”, bath, setting 5.
(13)
Spin out debris from samples: 10’, 14,000 RPM,
4ºC.
MEANWHILE….
(14)
Wash dynabeads for pre-clearing: 3X TE washpipet to resuspend, let sit on ice 3-5’, collect
beads, change wash. Resuspend last time in
dilution buffer. Wash 75L bead slurry per sample,
bring up in 75L, too.
(15)
Add soluble lysate to beads, keep cold pending
BCA (4ºC, shaker).
(16)
Remove 20L for INPUT (store at –20ºC),
(17)
Take 1L for BCA assay. Add to 1500L BCA
solution.
I use a 1:10 dilution of this 1L and do a 2L
and 4L reaction, and check the agreement of
those….
BCA ASSAY- As per Pierce protocol. NOTE: Takes 30’
incubation PLUS cooling, PLUS reading.
(18)
Finally….SET UP IP’s.
Use 500g (bare minimum, say your prayers)1000 g/ IP.
Magnet out beads before, keep remaining
chromatin after.
Add 5L (MyoD) antibody, O/N, 4ºC. Nutating.
NB: Some antibodies like to have some SDS around.
MyoD polyclonal (6975B) likes to have 0.05% SDS, or
1:400 of 20% stock. Also add this SDS to
subsequent IP buffers, until IP buffer 3, at the
same concentration for best results.
NEXT DAY……
(19)
Prepare some beads for recovering the IP by
washing them as before: 3 X 1mL TE, bring up in
dilution buffer. Prepare 30L dynabeads/IP.
(20)
Add BSA (1:10) and herring sperm/calf thymus
DNA (1:50) to dynabead blocking solution. Incubate
at 4ºC, nutating, 10’.
(21)
Add beads to IP, incubate 4ºC, nutating, 1 hr.
For the following washes, add 1mL, pipet to
resuspend, incubate 3-5’ on ice, collect beads, change
the wash. Also, once you get to TE wash #1, use
aerosol tips and treat the samples like PCR samples, as
far as contamination care goes.
Also, after the first TE, which will have no detergent
in it, the beads are considerably less adherent to the
wall of the tube, so aspirate with a pipetman to be
sure that if you suck something out, you can put it
back.
(22)
TE,
When
Collect
WASH BEADS: (IP Wash) #2, #2, #2, #3, #3, #3,
TE, TE.
in last TE wash, transfer to a fresh tube.
the beads.
(23)
ELUTE with 2 x 250L Elution buffer, 15’, 4ºC,
shaking.
(24)
Bring up the input sample volume to 500L with
Elution buffer.
(25)
Add 20L of 5M NaCl to elution volume, mix
well, and incubate at 65ºC, O/N (4-16hr.) to
decrosslink.
NEXT DAY….
(26)
Add to each sample: 10 L 0.5M EDTA, pH 8.0
20L 1.0M Tris, pH 6.5
5 L of Proteinase K (@50
mg/mL)
INCUBATE at 45-55ºC for 2 hrs.
(27)
CLEAN UP* the digestion mix with a QIAGEN PCR
Cleanup Kit.
*Add the reaction volume to 5X the volume of Qiagen
Buffer PB, mix well, and purify on a PCR purification
column. Wash as directed by kit. Elute in 40L-50L
of ChIP RESUSPENSION BUFFER (3mM Tris, pH 8.0, 0.1mM
EDTA).
(28)
Take the OD of input samples, and get ready to
set up the PCR’s. Store samples at –20ºC when not
in use. *You may not have enough IP sample to
detect an accurate OD. I usually start my ChIP
PCR’s with 2L of my elution per reaction and see
how it goes.
SOLUTIONS FOR CHROMATIN IP
PROTOCOL
10X FORMALDEHYDE FIXATION BUFFER
reagent
50mM HEPES, pH 8.0
1mM EDTA, pH 8.0
0.5mM EGTA
100mM NaCl
stock
1.0M
0.5M
0.5M
5.0M
volume (100mL)
5mL
200L
100L
2mL
*Add 37% Formaldehyde FRESH before use….
Buffer + 1.5 mL 37% Formaldehyde
Chromatin CELL WASH #2 BUFFER
reagent
stock
volume (500mL)
10mM Tris, pH 8.0
1.0M
5mL
10mM EDTA, pH 8.0
0.5M
10mL
0.5mM EGTA
0.5M
500L
0.25% Triton-X
20%
6.25mL
3.5 mL 10X
volume (100mL)
1mL
2mL
100L
1.25mL
*Add Sodium Butyrate FRESH before use, if doing an
acetylation-type IP…
10mM Butyrate
0.25M
4mL
20mL
Chromatin CELL WASH #3 BUFFER
reagent
stock
volume (100mL)
volume (500mL)
10mM Tris, pH 8.0
1.0M
1mL
5mL
1mM EDTA, pH 8.0
1mL
0.5mM EGTA
500L
200mM NaCl
20mL
0.5M
200L
0.5M
100L
5M
4mL
*Add Sodium Butyrate, as above, if needed.
4mL
20mL
ELUTION BUFFER
stock
(100mL)
1% SDS
0.1M NaHCO3
volume
20%
(1mol=84.01g)
0.84g
5mL
---ad
100 mL with H20
“DILUTION”/LYSIS BUFFER
reagent
stock
volume (100mL)
1.1% Triton-X
20%
5.6mL
4mM EDTA, pH 8.0
0.5M
800L
40mM Tris, pH 8.1
1.0M
4.0mL
300mM NaCl
5.0M
6.0mL
-------------------------------------------------------------------------Inhibitor Cocktail
7X
1:7 (14.29mL)
volume (2mL)
111L
16L
80L
120L
-------------------------286L
(from Boehringer pellet)
--------------------------------------------------------------------------------------------------*10mM Butyrate, if needed 0.25M
80L
4.0mL
+1.307mL H20
ad
100 mL with H20
2.0mL total
IP WASH BUFFER #2
reagent
stock
volume (200mL)
1% Triton-X
20%
10mL
2mM EDTA, pH 8.0
0.5M
800L
20mM Tris, pH 8.1
1.0M
4mL
500mM NaCl
5.0M
20mL
H20
---165mL
volume (100mL)
5mL
400L
2mL
10mL
82.6mL
**Add SDS as needed/preferred by the specific antibody
(MyoD IP- add SDS to 0.5%)
IP WASH BUFFER #3
reagent
volume (200mL)
stock
volume (100mL)
0.25M LiCl
5M
10mL
1% NP-40
20%
10mL
1% Na-Deoxycholate
20%
10mL
1mM EDTA, pH 8.0
0.5M
400L
10mM Tris, pH 8.1
1.0M
2mL
H20
---167.6mL
5mL
5mL
5mL
200L
1mL
83.8mL
Additional notes…
I check products on 5.5% polyacrylamide gels, 1X TBE,
5% glycerol. Run these at 60mAmps for 3 hours.
A typical PCR reaction looks like this:
2L IP sample
2L 10x PCR buffer
2L dNTP mix (10mM each)
2L DMSO
0.2L BSA (100X)
25pmol each primer per reaction
1.6L Mg++ (50mM)
0.33L Platinum Taq
0.1L dCTP
WHEN YOU’RE READY TO HARVESTCELLS:


BY SCRAPING
Warm DM, DME+0.5%HS+insulin+transferrin (25 mL
per condition), PBS
Prepare formaldehyde fix: Need 2.5 mL per
condition
For 5 mL, it should be 3.5 mL Fixation
buffer
1.5 mL 37%
Formaldehyde
SCRAPING PROTOCOL…





Dump the media from the cells from ONE CONDITION
into a conical tube and KEEP this media.
Take out sample for RNA - To keep some lysate for
eventual RNA analysis, scrape approximately 2
squares from the 15cm plate grid of cells in
remaining media (usually works out to be about
0.5mL). (spin down pellet, decant liquid, store at
-80ºC)
Add back same media gently to the plate(s) EXCEPT
the 0H high serum plate. To this plate, add back
25mL of warm, DM with insulin and transferrin but
no -estradiol.
Add fix- 2.5mL fix solution per plate, mix,
incubate on benchtop, RT, 30’.
Quench fix with 1.4mL of 2.5M Glycine per 25mL
media. Let sit for 3’. If staggered, put on ice
until last sample is done.
FOR THE REST OF THE WASH STEPS, it is better to do
them in the cold room and leave the plates incubating
there.
Use the same washes and wash times as for the
trypsinization protocol, EXCEPT after a wash/fix
step, just dump off the buffer, remove the last bit
of solution with a pipet, add the next wash, let sit
at 4ºC for 10’, then dump stuff….
After the last wash, scrape the cells with a cell
scraper, collect, pellet, decant sup. And proceed…..