ChIP

Overview
Chromatin Immunoprecipitation determines the in vivo chromatin binding sites of a transcription factor or other protein of interest. Use this protocol in conjunction with ChIP-chip and/or Gene-specific PCR.

Materials
''This protocol is in development. Need to add further info about reagents/equipment. ~ cmc 11:43, 22 August 2006 (EDT)''

11% Formaldehyde Solution
per 50 ml:                          (Final concentration)
 * 14.9 ml of 37% Formaldehyde         (11%)
 * 1 ml of 5M NaCl                     (0.1M)
 * 100 µl of 0.5 M EDTA, pH 8         (1 mM)
 * 50 µl of 0.5 M EGTA, pH 8          (0.5 mM)
 * 2.5 ml of 1M Hepes, pH 8           (50 mM)

2.5 M glycine (187.5 g/L)

 * Dissolve glycine in water with constant stirring
 * Don’t adjust pH

BSA/PBS Solution
per 100 mL:
 * 10 mL of 10x PBS
 * 500 mg of BSA
 * 90 mL of H20
 * Dissolve the BSA in H2O before adding the PBS.
 * Can make a liter, filter, aliquot by 200 mL, and store at 4°C. Good for months.

Lysis Buffer 1 (LB1)
per 100 ml:                        (final concentration)
 * 5 ml of 1M Hepes-KOH, pH 7.5	    (50 mM)
 * 2.8 ml of 5M NaCl		    (140 mM)
 * 0.2 ml of 0.5M EDTA		    (1 mM)
 * 10 ml of glycerol		    (10%)
 * 5 ml of 10% NP-40		    (0.5%)
 * 0.25 ml of Triton X-100		    (0.25%)

Lysis Buffer 2 (LB2)
per 100 ml:			(final concentration)
 * 4 ml of 5M NaCl			(200 mM)
 * 0.2 ml of 0.5M EDTA		(1 mM)
 * 0.1 ml of 0.5M EGTA		(0.5 mM)
 * 1.0 ml of 1M Tris pH 7.5	(10 mM)

Lysis Buffer 3 (LB3)
per 100 ml:			(final concentration)
 * 0.2 ml of 0.5M EDTA		(1 mM)
 * 0.1 ml of 0.5M EGTA		(0.5 mM)
 * 1.0 ml of 1M Tris-HCl, pH7.5	(10 mM)
 * 2 mL of 5M NaCl			(100 mM)
 * 1 mL of 10% Na-Deoxycholate	(0.1%)
 * 500 mg of N-lauroyl sarcosine	(0.5%)

Wash buffer (RIPA buffer)
per 100ml:			    (final concentration)
 * 5ml of 1M Hepes, pH 7.6		   (50 mM)
 * 200µL of 0.5M EDTA		   (1 mM)
 * 7 ml of 10% DOC (Na deoxycholate) (0.7%)
 * 10 ml of 10% NP-40 (IPGEL)	   (1%)
 * 10 ml of 5M LiCl or 2.12g powder	   (0.5 M)

Elution buffer

 * 50mM Tris pH8
 * 1mM EDTA
 * 1% SDS

Oligos for blunt end ligation

 * oJW102: 5’-GCGGTGACCCGGGAGATCTGAATTC
 * oJW103: 5’-GAATTCAGATC
 * Final buffer/concentrations for annealing:
 * 50 mM Tris pH 7.5
 * 50 mM NaCl
 * 17.5 µM each oligo
 * After mixing, boil 5 minutes, and anneal slowly from a 100°C heat block to 25°C, then cool to 4°C overnight, aliquot, and freeze.

I. Cell cross-linking

 * Use 5x107 – 1x108 cells (70-80% confluency for adhesion cells of 8-12 15 cm2 plates or 175 cm2 flasks) for each ChIP reaction.


 * Adherent cells:
 * Add 1/10 volume of fresh 11% formaldehyde solution to plates.
 * Swirl plates briefly and let them sit at RT for 10 min.
 * Add 1/20 volume of 2.5 M glycine to plates to quench formaldehyde.
 * Rinse cells twice with 5 ml 1X PBS. Harvest cells using silicon scraper.
 * Spin cells at 4k for 10’ at 4°C.
 * Transfer cells to 15ml conical tubes and spin 4k 10’ at 4°C.
 * Flash freeze cells in liquid nitrogen and store pellets at –80 °C.
 * Suspension cells:
 * Add 1/10 volume of fresh 11% formaldehyde solution to flasks.
 * Swirl flasks briefly and let them sit at RT for 20 min.
 * Add 1/20 volume of 2.5 M glycine to flasks to quench formaldehyde.
 * Spin cells at 2500rpm for 10’ at 4°C.
 * Rinse cells with 100ml 1x PBS and spin cells at 2500rpm for 10’ at 4°C.
 * Rinse cells with 10ml 1x PBS and transfer to 15ml conical tubes and spin 4k 10’ at 4°C.
 * Flash freeze cells in liquid nitrogen and store pellets at –80 °C.

II.	Preblock and binding of antibody to magnetic beads

 * Keep beads on ice for all steps (or work at 4°C).
 * 1) wash 100 µL of Protein G Dynal magnetic beads (per reaction) in 1 ml fresh BSA/PBS
 * 2) collect the beads with magnet wash beads in 1.5 ml BSA/PBS 2 times, collect the beads with the magnet.
 * 3) Add 6-10 µg of Ab + 250µL of PBS/BSA.
 * 4) incubate 4 hr to O.N. on a rotating platform at 4°C.
 * 5) wash beads 3 times in 1.5 ml PBS/BSA.
 * 6) Resuspend in 100µL PBS/BSA.

III. Cell Sonication

 * Note: Add protease inhibitors to all lysis buffers before use. If using a protease inhibitor cocktail tablet from Complete, dissolve one tablet in 2 ml H2O for 25x solution.  Use 400uL per 10mL of buffer. Store in aliquots at -20°C.
 * 1)  Resuspend each tube of cells in 5-10 ml of Lysis buffer I. Rock at 4°C for 10’.  Then spin at 2500rpm in a table top centrifuge, 3 min at 4°C.
 * 2) Resuspend each tube of cells in 5-10 ml of  buffer 2.  Rock gently at 4°C for 5 min.  Pellet nuclei in tabletop centrifuge by spinning at 2500  rpm, 3 min at 4°C.
 * 3) Resuspend pellet in each tube in 3 ml buffer 3 and put into 15mL conical tube cut off at the 6.5mL mark.
 * 4) Sonicate the suspension using the automatic sonicator.
 * 5) * Appropriate sonication time needs to be determined for each cell type. For example, HepG2 cells: 6 cycles of 30 seconds with 1 minute intervals at power 7.0 (~40 watts).
 * 6) Add 1/10 volume of 10% Triton X-100. Transfer to 1.5 ml centrifuge tubes.  Spin out debris 14K, 4°C, 10 min.
 * 7)  If you are using one tube of cells for more than one ChIP then you need to add more LB3 and 10% Triton X-100 so that the volume ends up being 3 mL of cells in LB3 with 300uL of Triton per ChIP.
 * 8) Save 50 µL (or at least 1/50 vol) of cell lysate from each sample as input control. Store at -20°C.
 * 9) * Don't forget this!!

IV. Chromatin Immunoprecipitation

 * 1) Add 100 µL Ab prebound Dynal magnetic beads from step III.
 * 2) Rock 4°C 6 hrs to O/N.

V. Washing, eluting, and reverse cross-linking

 * Check that you have a waterbath or incubator at 65°C.
 * Work in the cold room until step 6.
 * 1) Transfer IP reactions to centrifuge tubes
 * 2) Use the magnetic stand to precipitate the beads.
 * 3) Wash 4-8 times with 1 mL wash buffer (Ripa buffer)
 * 4) * 4 washes are sufficient for most antibodies.
 * 5) Wash once with 1 ml TE-plus-50 mM NaCl or 1 mL TBS.
 * 6) Spin 3k for 2-3 min and aspirate any residual TE/TBS.
 * 7) Add 200 µl of elution buffer.
 * 8) Elute DNA-protein complexes from beads at 65°C for 10-15 min with brief vortexing every 2 min.
 * 9) Spin down beads 14k for 1 min. Transfer all 200 µl of supernatant to a clean 1.5 mL tube.
 * 10) * Alternatively: pulse samples to pull beads off the lid, but leave beads in until after the reverse x-link incubation.
 * 11) Reverse x-link at 65°C. Min 6 hours, up to O/N.
 * 12)  Thaw input (wce) samples from step III(6), add 3 vol of elution buffer and reverse x-link in parallel with IP samples, 6 hrs - o/n.

VI. RNase, Proteinase K

 * 1) Remove beads from IP samples, if you didn't earlier.
 * 2) Add 1 vol of TE to IP and input (wce) fraction.
 * 3) Add RNase A so final is 0.2µg/µL (~8 µL/400 µL rxn).  Incubate 37°C 1-2hr.
 * 4) Add proteinase K so final is 0.2µg/µL (~4 µL/400 µL rxn). Incubate 55°C 1-2hr.
 * 5) Extract once w/ 1 vol of phenol.
 * 6) Extract once w/ 1 vol of phenol:chl:IA (made by mixing 1 vol of phenol w/ 1 vol of Chloroform:isoamyl alcohol).
 * 7) extract once w/ 1 vol of chl:IA.
 * 8) * Alternatively: extract 1x w/ 1 vol phenol:chl:IA using phaselock tubes
 * 9) add 20 µg (1 µL) of glycogen.
 * 10) Add 5M NaCl so final is 0.2M (10 µL/250 µL rxn).
 * 11) Add 2X volume of EtOH.
 * 12) * Optional: incubate samples at -20°C for 10 min.
 * 13) Precipitate DNA with a 10 min, 14K spin at 4°C. Wash pellet with 500 µL 75% EtOH.
 * 14) Dry and resuspend pellets in 60 µL 10mM Tris HCl pH 8.
 * 15) Normalize the input/wce fraction to 100 ng/µL using the Nanodrop.

VII. T4 DNA Polymerase Fill-in

 * 1) To 60 µL of IP sample, and 200 ng (=2 µL) of the normalized input (wce) diluted to 60 µL, add:
 * 2) *10 µL of 10X T4 DNA pol buffer (or NEB Buffer 2)
 * 3) * 0.5 µL of BSA (NEB 10mg/ml)
 * 4) * 0.4 µL of 25mM dNTP mix
 * 5) * 0.2 µL of T4 DNA pol (NEB 3U/µL)
 * 6) * 28.9 µL of H2O
 * 7) * final volume =100uL (add 39.8 uL of mix per sample.)
 * 8) Incubate 12°C 20 min in water bath.
 * 9) Add 12 µl 3 M NaOAc and 1.0 µL  glycogen.
 * 10) Extract 1x with 120 µL phenol:chl:IA.
 * 11) Precipitate with 300 µL EtOH.
 * 12) Spin and wash with 500 µL 75% EtOH.
 * 13) Dry pellet and resuspend in 25 µL H2O.

VIII. Blunt-end ligation

 * 1)  Make at 4°C, 25 µL ligase mix per reaction:
 * 2) * 7.8 µL of H2O
 * 3) * 10 µL of 5X ligase buffer (Gibco)
 * 4) * 6.7 µL of annealed linkers (thaw at 4°C)
 * 5) * 0.5 µL of T4 DNA ligase
 * 6) add mix to 25 µL of sample.
 * 7) incubate 12°C O/N, cover with foil.
 * 8) Next day add 6µL of 3M NaOAc and 150 µL EtOH. (Optional incubation at -20°C.)
 * 9) Spin and wash with 200 µL 75% EtOH.
 * 10) Dry and resuspend pellet in 40 µL PCR reaction mix below.

IX. Ligation mediated PCR (LM-PCR)

 * for PCR: use pellets from blunt ligation.
 * 1) Make PCR mix per rxn:
 * 2) * 35 µL of H2O
 * 3) * 4 µL of 10X Thermopol buffer
 * 4) * 0.5 µL of 25 mM dNTP mix
 * 5) * 0.5 µL of 100 µM oligo oJW102
 * 6) Dissolve pellet in mix above and start PCR program CHIPCHIP:
 * 7) * 55°C, 4’; 72°C, 3’; 95°C, 2’; 95°C, 30’’; 60°C, 30’’; 72°C, 1’; goto step 4 14 times; 72°C, 5’; 4°C, hold.
 * 8) During step 1 (55°C, 4’) add TAQ mix:
 * 9) * 8 µL of H2O
 * 10) * 1 µL of 10X ThermoPol buffer
 * 11) * 0.5 µL of TAQ (5U/µL)(Gibco)
 * 12) Dilute PCR product in 450uL of 10 mM Tris, pH 8.0.
 * 13) Take 10uL of diluted product and PCR again using PCR mix per rxn:
 * 14) * 25 µL of H2O
 * 15) * 4 µL of 10X Thermopol buffer
 * 16) * 0.5 µL of 25 mM dNTP mix
 * 17) * 0.5 µL of 100 µM oligo oJW102
 * 18) Start PCR program:
 * 19) * 55°C, 4’; 72°C, 3’; 95°C, 2’; 95°C, 30’’; 60°C, 30’’; 72°C, 1’; goto step 4 24 times; 72°C, 5’; 4°C, hold.
 * 20) During step 1 (55°C, 4’) add TAQ mix:
 * 21) * 8 µL of H2O
 * 22) * 1 µL of 10X ThermoPol buffer
 * 23) * 0.5 µL of TAQ (5U/µL)(Gibco)
 * 24) Clean up DNA using Qiagen kit or precipitation by adding 25µL of 7.5M NH4OAc and 300µL EtOH. (Optional incubation at -20°C.) Spin and wash pellet with 500 µL 75% EtOH. Redissolve in 50 µL H2O.
 * 25)   Normalize [DNA] to 100 ng/µL.

Checkpoints

 * 1) Sonication Fragment Size Verification
 * 2) * After sonication, the spread of size fragments can be checked by running 1 µL of input/wce control DNA on a 1% agarose gel. Spread should extend no higher than 2 kb.
 * 3) Product Size and Amount Verification
 * 4) * Remove 2 µL of PCR product and run it out on 2% agarose gel. Product sizes should be around 250-350 bp.  Amount of IP PCR product should be roughly equal to input PCR product.  Check the concentration using the Nanodrop, expecting ~50-100 ng/µL.

Detection

 * Continue with ChIP-chip or Gene-specific PCR to detect global or specific chromatin binding sites for your protein of interest.

Contacts

 * cmc
 * odom