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REVISED CHROMATIN IP PROTOCOL

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REVISED CHROMATIN IP PROTOCOL Powered By Docstoc
					  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 wash-
  pipet 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)     WASH BEADS: (IP Wash) #2, #2, #2, #3, #3, #3,
    TE,   TE, TE.
   When   in last TE wash, transfer to a fresh tube.
Collect   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              stock            volume (100mL)
50mM HEPES, pH 8.0        1.0M              5mL
1mM EDTA, pH 8.0     0.5M             200L
0.5mM EGTA           0.5M             100L
100mM NaCl           5.0M              2mL

*Add 37% Formaldehyde FRESH before use….      3.5 mL 10X
Buffer + 1.5 mL 37% Formaldehyde


Chromatin CELL WASH #2 BUFFER
reagent              stock            volume (100mL)
     volume (500mL)
10mM Tris, pH 8.0    1.0M              1mL
5mL
10mM EDTA, pH 8.0    0.5M              2mL
10mL
0.5mM EGTA           0.5M             100L
500L
0.25% Triton-X            20%                1.25mL
6.25mL

*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      0.5M            200L
1mL
0.5mM EGTA             0.5M            100L
500L
200mM NaCl             5M                    4mL
20mL

*Add Sodium Butyrate, as above, if needed.
4mL               20mL




ELUTION BUFFER
                              stock          volume
(100mL)
1% SDS                          20%                     5mL
0.1M NaHCO3          (1mol=84.01g)                   ----
                  0.84g
                                                       ad
100 mL with H20

“DILUTION”/LYSIS BUFFER
reagent              stock            volume (2mL)
    volume (100mL)
1.1% Triton-X             20%                111L
5.6mL
4mM EDTA, pH 8.0     0.5M               16L
800L
40mM Tris, pH 8.1    1.0M               80L
4.0mL
300mM NaCl           5.0M             120L
6.0mL
----------------------------------    ----------
    --------------------------        -----------------
----------------
Inhibitor Cocktail         7X                286L
1:7 (14.29mL)
(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 (100mL)
     volume (200mL)
1% Triton-X           20%                     5mL
10mL
2mM EDTA, pH 8.0              0.5M                  400L
             800L
20mM Tris, pH 8.1          1.0M               2mL
4mL
500mM NaCl            5.0M             10mL
20mL
H20                 ----               82.6mL
165mL

**Add SDS as needed/preferred by the specific antibody
(MyoD IP- add SDS to 0.5%)




IP WASH BUFFER #3
reagent              stock            volume (100mL)
    volume (200mL)
0.25M LiCl            5M                     5mL
10mL
1% NP-40          20%                 5mL
10mL
1% Na-Deoxycholate         20%                      5mL
10mL
1mM EDTA, pH 8.0      0.5M                  200L
400L
10mM Tris, pH 8.1     1.0M                   1mL
2mL
H20                 ----                    83.8mL
167.6mL




             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…..

				
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