User Guide

User Guide

User Guide

Affymetrix

®

Chromatin

Immunoprecipitation Assay

Protocol

P/N 702238 Rev. 4

For research use only.

Not for use in diagnostic procedures.

Trademarks

Affymetrix ® , Axiom ® , Command Console ® , CytoScan ™ , DMET ™ , GeneAtlas ® , GeneChip ® , GeneChip-compatible ™ , GeneTitan ® ,

Genotyping Console ™ , myDesign ™ , NetAffx ® , OncoScan ™ , Powered by Affymetrix ™ , PrimeView ™ , Procarta ® , and QuantiGene ® are trademarks or registered trademarks of Affymetrix, Inc. USB, the logo design, and PrepEase are registered trademarks of

USB Corporation. All other trademarks are the property of their respective owners.

Limited License

Subject to the Affymetrix terms and conditions that govern your use of Affymetrix products, Affymetrix grants you a nonexclusive, non-transferable, non-sublicensable license to use this Affymetrix product only in accordance with the manual and written instructions provided by Affymetrix. You understand and agree that except as expressly set forth in the Affymetrix terms and conditions, that no right or license to any patent or other intellectual property owned or licensable by Affymetrix is conveyed or implied by this Affymetrix product. In particular, no right or license is conveyed or implied to use this Affymetrix product in combination with a product not provided, licensed or specifically recommended by Affymetrix for such use.

Patents

Arrays: Products may be covered by one or more of the following patents: U.S. Patent Nos. 5,445,934; 5,744,305; 5,945,334;

6,140,044; 6,399,365; 6,551,817; 6,733,977; 7,629,164; 7,790,389 and D430,024 and other U.S. or foreign patents. Products are manufactured and sold under license from OGT under 5,700,637.

PrepEase products are covered under European Patent EP 0496822 and US Patent 6,428,703.

Copyright

© Affymetrix Inc. All rights reserved.

Contents

Chapter 1

Chapter 2

Chapter 3

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4

Chromatin Immunoprecipitation Assay Protocol Optimization . . . . . . . . . . . . . . . . . . . . . . . .4

Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6

Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

Miscellaneous Reagents and Supplies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

Chromatin Immunoprecipitation Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Procedure A: Prepare Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

Procedure B: Fix Cells, Lyse, and Sonicate Whole Cell Extracts . . . . . . . . . . . . . . . . . . . . . . .11

Adherent Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

Suspension Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11

Wash Cell Pellet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

Procedure C: Check Sonication Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12

Procedure D: Incubate With Specific Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13

Procedure E: Immunoprecipitate and Wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

Procedure F: Reverse Crosslinks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Procedure G: Cleanup De-crosslinked Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15

Procedure H: PCR Amplify Immunoprecipitated DNA Targets . . . . . . . . . . . . . . . . . . . . . . .15

Procedure I: Fragment Amplified Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19

Procedure J: Label Fragmented Double-Stranded DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . .20

Hybridization and Array Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Procedure A: Hybridize Labeled Target on the Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

Procedure B: Array Wash, Stain and Scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22

Appendix A Cleanup of Double-Stranded DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Cleanup of Double-Stranded DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23

Brief Protocol for Concentration, Desalination and/or Removal of Enzymes . . . . . . . . . . .23

Brief Protocol for PCR Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23

Brief Protocol for DNA Purification from Chromatin Immunoprecipitation (ChIP) Assay . .24

Appendix B Contact Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

1

Overview

Introduction

The Affymetrix

®

Chromatin Immunoprecipitation (ChIP) Assay is designed to generate double-stranded labeled DNA targets that identify sites of protein-DNA interactions or chromatin modifications on a genome-wide scale. This assay has been designed specifically for use with Affymetrix GeneChip® Tiling

Arrays for ChIP-on-chip studies in order to study transcription factor binding sites, histone protein modifications, and other chromatin-protein interactions.

ChIP experiments can be used as a powerful tool to complement RNA transcription studies because they enable researchers to study the DNA-protein interactions that regulate gene expression. Following the protocol, cells are first fixed with formaldehyde to crosslink DNA to any associated proteins. The cells are then lysed and DNA is sheared into smaller fragments using sonication. Protein-DNA complexes are then immunoprecipitated with an antibody directed against the specific protein of interest. Following the immunoprecipitation, crosslinking is reversed, samples are protease-treated and the purified DNA sample is amplified using a random-primed PCR method. Subsequently, targets are fragmented and labeled to hybridize onto GeneChip ® Tiling Arrays. By comparing the hybridization signals generated by an immunoprecipitated sample versus an antibody-negative or non-specific antibody control, the regions of chromatin-protein interaction can be identified.

Studies were performed at Affymetrix to evaluate the robustness and sensitivity of the ChIP assay; however, because of the variability associated with the quality and affinity of various antibodies against their intended targets, results may vary from one antibody to the next. The procedure outlined in this protocol describes all the necessary steps and reagents for fixing cells, fragmenting chromatin, immunoprecipitating sheared chromatin, amplifying and labeling precipitated DNA.

We would like to acknowledge Mark Biggin and Xiao-Yong Li of the Lawrence Berkeley National Lab for sharing their modifications to the ChIP protocol. We have incorporated their improvements to the amplification step (

on page 15 ) with their approval.

Chromatin Immunoprecipitation Assay Protocol Optimization

This protocol has been developed for use with GeneChip

®

Tiling Arrays. Exact protocol conditions will require optimization by each user due to the variability inherent in:

Experimental Biology: cell types, proteins of interest, antibodies

Assay conditions: DNA fragmentation, PCR conditions

To ensure success with this protocol, it is critical that users optimize the following steps in the ChIP protocol prior to performing microarray hybridizations. Additional information on the optimization steps are available throughout this protocol.

1. Sonication conditions of fixed cells.

Some cells are resistant to sonication treatment. Micrococcal nuclease treatment may improve DNA shearing for some cell lines.

2. Antibody Qualification.

Antibodies should be qualified for use with chromatin immunoprecipitation experiments. ChIP qualification information is available from www.chiponchip.org

or directly from antibody vendors.

3. Antibody Titration.

Antibody affinities and avidities can vary, so the amount of antibody may need to be titrated to achieve optimal sample enrichment.

4. PCR amplification of immunoprecipitated DNA.

The optimal number of PCR cycles may require optimization to avoid saturation and ensure that the

IP enrichment is maintained.

Chapter 1 | Overview

5

5. QPCR Positive Control.

A QPCR control is recommended to test ChIP conditions. This test requires a known protein binding to a known DNA sequence. After performing ChIP with an antibody to the known protein, QPCR is used to verify that the known DNA binding elements are enriched in experimental vs. negative control samples. This QPCR test can also be used to ensure that the enrichment of experimental samples vs. control samples is maintained after IP column clean up.

Figure 1.1 Chromatin Immunoprecipitation Assay Schematic

Fix cells to crosslink DNA to protein

Sonicate to lyse cells and shear chromatin

Take small aliquot to decrosslink and check sonication efficiency

Immunoprecipitate main sample with selected antibody

Couple to

Protein-A beads and wash to purify IP'd DNA

Decrosslink and proteinase treat

IP'd DNA

Sample Cleanup

Linear

Amplification

PCR amplify and incorporate dUTP

Fragment and label amplified

DNA target

Clean up

IP'd DNA

Random

Primer/Adapter

Sequenase, dNTPs

Adapter

5'

3'

5'

3'

5'

3'

Fragmentation

U

U

3'

5'

Terminal Labeling

+

3'

3'

5 '

Adapter Primer, dNTP + dUTP,

Taq polymerase

5 '

5'

3'

U

U

U

U

Uracil DNA Glycosylase

APE 1

3'

5'

Terminal Deoxynucleotidyl

Transferase DLR (Biotin-labeled)

+

Day 1

Day 2

Day 3

Hybridize arrays

Hybridization

Washing/Staining

Scanning

Legend

Proteins Antibody

SAPE Biotinylated anti-streptavidin antibody

Protein-A beads DNA

Day 4

Chapter 1 | Overview

6

Materials

Table 1.1 Materials Required

Material Source Part Number

Formaldehyde Solution (37%), 500 mL

Glycine, 1 kg

Phosphate Buffered Saline (PBS) pH 7.4 (1X), liquid

IGEPAL ® CA-630

Phenylmethanesulfonyl Fluoride Solution (PMSF), 250 mL

Microccocal Nuclease (MNase) (Optional)

EGTA (optional)

Protease Inhibitor Tablet

Decrosslink and Check Sonication Efficiency

Proteinase K

Sigma-Aldrich

Sigma-Aldrich

Various

Sigma-Aldrich

Sigma-Aldrich

USB

Sigma-Aldrich

Roche

F8775

50046

9036-19-5

93482

70196Y

E3889-100G

11873580001

LiCl (8M), 500 mL

Glycogen

Immunoprecipitation

Triton-X100 (non-ionic viscous liquid)

New England BioLabs P8102S

Sigma-Aldrich

Roche

Protein A Sepharose ™ CL-4B

Antibody

NOTE: Antibody should be qualified for chromatin immunoprecipitation. See www.chiponchip.org for a list of qualified antibodies.

Roche

Amersham

Various

PCR Amplification

Sequenase ™ Version 2.0 DNA Polymerase USB

Primer A: 200 µM GTTTCCCAGTCACGGTC(N)

9

Various

Primer B: 100 µM GTTTCCCAGTCACGGTC Various

L7026

10901393001

10789704001

17-0963-03

70775Y

HPLC purified

Taq Polymerase 5 U/µL

10X PCR Buffer dATP 100 mM dCTP 100 mM

Various

Various

Various

Various dGTP 100 mM dTTP 100 mM dUTP 100 mM

BSA 20 mg/mL

DTT 0.1M

Various

Various

Various

Various

Various

Chapter 1 | Overview

7

Table 1.1 Materials Required (Continued)

Material

Wash Buffer

Tris-HCl

EDTA

SDS, 100g

NaCl

Deoxycholate (sodium salt), 100g

MgCl

2

, 1M

CaCl

2

, 1M

Fragmentation and Labeling

Uracil-DNA Glycosylase (UDG) (2 U/µL)

Human Apurinic/Apyrimidinic Endonuclease 1 (APE 1)

(Includes 10X APE 1 Reaction Buffer)

Terminal Deoxynucleotidyl Transferase (rTdT), Recombinant, (30 U/µL)

(5X TdT buffer included)

DNA Labeling Reagent, DLR, 10 mM

DNA Cleanup

PrepEase DNA Clean-Up Kit

Hybridization, Stain and Wash

GeneChip ® Hybridization, Wash, and Stain Kit

Control Oligonucleotide B2, 3nM

Source

Various

Various

Sigma-Aldrich

Various

Sigma-Aldrich

Various

Sigma-Aldrich

USB

USB

USB

USB

USB

Affymetrix

Affymetrix

Part Number

71725

D6750

21115

71960

78454

72033

79015

78758

900720

900301

Buffers

Table 1.2 Buffers

Lysis Buffer (Store at 4°C)

10 mM Tris-HCl (made from stock 1M Tris-HCl pH 7.5)

10 mM NaCl

3 mM MgCl

2

0.5% IGEPAL

1 mM PMSF (add fresh)

Pre-IP Dilution Buffer (Store at RT)

10 mM Tris-HCl (made from stock 1M Tris-HCl pH 7.5)

10 mM NaCl

3 mM MgCl

2

1 mM CaCl

2

4% IGEPAL

1 mM PMSF (add fresh)

IP Dilution Buffer (Store at RT without protease inhibitors)

20 mM Tris-HCl (made from stock 1M Tris-HCl pH 8)

2 mM EDTA

1% Triton X-100

150 mM NaCl

Protease Inhibitor Stock (add fresh)

Protease Inhibitor Stock

Prepare a 25X stock by dissolving 1 protease inhibitor tablet in 2 mL of nuclease-free water

ChIP Wash 1 (Store at RT)

20 mM Tris-HCl (made from stock 1M Tris-HCl pH 8)

2 mM EDTA

1% Triton X-100

150 mM NaCl

1 mM PMSF (add fresh)

ChIP Wash 2 (Store at RT)

20 mM Tris-HCl (made from stock 1M Tris-HCl pH 8)

2 mM EDTA

1% Triton X-100

0.1% SDS

500 mM NaCl

1 mM PMSF (add fresh)

ChIP Wash 3 (Store at RT)

10 mM Tris-HCl (made from stock 1M Tris-HCl pH 8)

1 mM EDTA

0.25M LiCl

0.5% IGEPAL

0.5% Deoxycholate (sodium salt)

Elution Buffer

25 mM Tris-HCl (made from stock 1M Tris-HCl pH 7.5)

10 mM EDTA

0.5% SDS

Chapter 1 | Overview

8

Chapter 1 | Overview

9

Miscellaneous Reagents and Supplies

Table 1.3 Miscellaneous Reagents and Supplies

Material

Miscellaneous Reagents

Supplier

Absolute ethanol

RNA-6000 Nano LabChip Kit

Gel-Shift Assay (Optional)

Novex XCell SureLock ™ Mini-Cell *

TBE Gel, 4-20%,10 mm, 12 well*

5X Sucrose Gel Loading Dye

10X TBE Buffer

SYBR ® Gold

10 bp DNA ladder and 100 bp DNA ladder

ImmunoPure NeutrAvidin

PBS, pH 7.2

Miscellaneous Supplies

1-2% Agarose Gells

1.5 mL RNase-free Microfuge Tubes*

1.5 mL Non-stick RNase-free Microfuge Tubes*

0.2 mL MicroAmp Reaction Tubes (8 tubes/strip)*

MicroAmp Caps for 8 Strip Tubes

Pipette for 25 mL*

Pipet-Aid*

Gold Shield Chemical Co.

Agilent

Invitrogen

Invitrogen

Amresco

Cambrex

Invitrogen

Invitrogen

Pierce

Invitrogen

Various

Ambion

Ambion

Applied Biosystems

Applied Biosystems

VWR

VWR

Dolphin-nose Tubes

SpinX Columns

MicroSpin ™ S-300 HR Columns

Instruments

Costar (Corning)

Costar (Corning)

GE Healthcare

Rotating Benchtop Platforms

Branson Sonifier ® S-450D

Double Step Micro Tip Assembly

NanoDrop ® ND-1000*

GeneChip ® Hybridization Oven 640

Eppendorf Centrifuge*

Various

Branson Ultrasonics

Branson Ultrasonics

Nanodrop Technologies

Affymetrix

Eppendorf

Refrigerated Centrifuge with swing bucket rotor Various

Tube-Strip Picofuge ™ Stratagene

GeneChip ® Fluidics Station 450 or 400 Affymetrix

Part Number

5065-4476

EI0001

EC62252

E-274

50843

S-11494

10821-015 and 15628-019

31000

20012-027

12400

12450

N801-0580

N801-0535

53283-710

53498-103

3213

8163

27-5130-01

101-063-590

101-063-212

ND-1000

8001318

5417C

400540

00-0079

Chapter 1 | Overview

10

Table 1.3 Miscellaneous Reagents and Supplies (Continued)

Material Supplier

GeneChip ® Scanner 3000 7G Affymetrix

GeneChip ® Autoloader (Optional)

ABI GeneAmp PCR System 9700*

Bioanalyzer 2100

Heating Block*

Pipette for 0.1 to 2 µL*

Pipette for 2 to 20 µL*

Pipette for 20 to 200 µL*

Pipette for 100 to 1,000 µL*

Affymetrix

Applied Biosystems

Agilent

VWR

Rainin

Rainin

Rainin

Rainin

* Or equivalent instrument/supplies.

Part Number

00-0073

90-0351

N/A

G2940CA

13259-030

L-2

L-20

L-200

L-1000

2

Chromatin Immunoprecipitation Assay

Procedure A: Prepare Cells

1. Grow enough cells for the number of immunoprecipitation (IP) reactions to be performed (usually

5 x 10 7 cells per IP for suspension cells, depending on IP efficiency). Prepare enough cells for two IP reactions. An antibody-minus (Ab- or mock IP) or non-specific IgG is recommended as a negative control using the same number of cells as the IP condition. The Ab- target would be treated identically to the experimental sample to serve as the “Control” group in the downstream two-sample analysis.

2. Use ~ 0.5 - 2 x 10

2 x 10 8 cells.

8 cells per IP. For example, grow 200 mL of 1 x 10 6 cells/mL for a total of

Procedure B: Fix Cells, Lyse, and Sonicate Whole Cell Extracts

DAY 1

NOTE: Centrifugation steps involving cells are best performed with a swing-bucket type rotor.

Adherent Cells

NOTE: End users may optimize the sequence of fixing and harvesting cells to minimize the degree to which cell physiology is disrupted.

1. Add formaldehyde to the culture flask to a final concentration of 1% and incubate in a fume hood for

10 minutes.

2. Add 1/20 volume of 2.5 M glycine and incubate at room temperature (RT) for 5 minutes with gentle mixing.

3. Pour off formaldehyde media into an appropriate waste container and add enough ice-cold 1X PBS to cover the bottom of the flask to wash cells. Pour off PBS into a formaldehyde waste container and add enough PBS to cover bottom of flask.

4. Using a cell scraper, scrape off cells to re-suspend and check flask with microscope to ensure that most cells are re-suspended.

5. From here, go to

Step 1 of the

Wash Cell Pellet

section below.

Suspension Cells

1. Fix cells by adding formaldehyde to a final concentration of 1% (add 5.5 mL of 37% formaldehyde to 200 mL of culture medium).

2. Incubate at room temperature (RT) in fume hood for 10 minutes, gently swirl 200 mL culture or invert tube containing 20 mL of adherent cells occasionally to mix cells.

3. Add 1/20 volume 2.5 M glycine and incubate at RT 5 minutes with gentle mixing to quench formaldehyde reaction. Perform remaining steps on ice.

4. Pellet cells at 4°C, (300-500g), 4 minutes and discard supernatant in formaldehyde waste.

Chapter 2 | Chromatin Immunoprecipitation Assay

12

Wash Cell Pellet

1. Wash pellet with 10 mL ice-cold 1X PBS to resuspend cells, and transfer to 15 mL tube.

2. Pellet cells at 4°C, (300-500g), 4 minutes and discard supernatant and repeat wash with ice-cold

1X PBS once.

3. Wash the pellet 3 times with 10 mL Lysis Buffer with fresh PMSF. Pellet cells at (300-500g)

5 minutes between washes.

4. Discard supernatant and proceed to the next step or flash freeze pellet and store at –80°C.

5. Resuspend the pellet in 1 mL pre-IP dilution buffer (add 60 μL PMSF) and bring final reaction volume to 1.5 mL with pre-IP dilution buffer.

6. Add to the tube:

Table 2.1

Component

100 mM PMSF

25X Protease Inhibitor Stock

Pre-IP Dilution Buffer

20% SDS

5 M NaCl

Nuclease-free Water

Final Sample Volume Before Sonication

*

if using optional MNase, see details on page 13 .

Volume for 1 Rxn

40 µL

100 µL

460 µL

100 µL

80 µL

220 µL *

2.5 mL

7. Sonicate sample to lyse cells and shear DNA to 100-1000 bp fragments. Some cell types (e.g., Jurkat) may require optional MNase treatment. See

page 13

for details.

NOTE: Optimized shearing conditions are cell-type and instrument dependent. It is recommended that conditions are optimized with a single sample prior to scaling up procedures to multiple samples. Best sonication conditions at Affymetrix were achieved with a Branson Sonifier 450D (using a double-step microtip) set at 60% duty, 50% amplitude, 1 minute pulses with 1 minute rest. Both pulsing and resting steps were performed in an ice bath, 8 to 10 pulses total for HL-60 cells. Number of pulses may be dependent on cell density as well as cell type.

8. Aliquot the sonicated samples into two 1.5 mL microcentrifuge tubes, then microcentrifuge

14,000 rpm 10 minutes at 4°C to remove cellular debris.

9. Pool supernatants (from

Step 8 ) in a 15 mL conical tube.

10. The sonication efficiency can be checked by taking an aliquot (100 μL) of this supernatant, decrosslinking it (see Procedure C, below), and running the de-crosslinked DNA on a 1-2% agarose gel.

11. Divide the samples into aliquots equivalent to ~ 5 x 10 7

cells (1 IP), flash freeze and store at –80°C for later use or take straight through the IP.

Procedure C: Check Sonication Efficiency

1. Add 100 μL 10 mM Tris pH 8.0 to the 100 μL aliquot taken from the sonicated samples.

2. Add 2 μL Proteinase K (20 mg/mL) and mix well by vortexing.

3. Incubate 42°C for 2 hours, then 65°C for 6 hours to overnight (This step can be performed in a thermocycler.)

Chapter 2 | Chromatin Immunoprecipitation Assay

13

4. Clean-up using USB PrepEase DNA Clean-Up Kit (see

Cleanup of Double-Stranded DNA

on page 23 ).

5. Load 100-500 ng of purified DNA sample on an agarose gel to check sonication efficiency. Typically, sheared DNA size ranges from 100-4000 bp, with the average size fragment between 200-1000 bp.

Figure 2.1 (A) Sheared DNA from HL-60 cells following 8 sonication pulses show the optimal size range for immunoprecipitation (~200-1000 bp with the majority of DNA fragments between 300-500 bp). Certain cell types may be more resistant to shearing by sonication and would require treatment with Micrococcal nuclease (MNase) to fragment chromatin. (B) Jurkat cells after 15 pulses of sonication show little fragmentation of crosslinked chromatin. (C) Fragmentation of Jurkat chromatin is achieved with MNase treatment. MNase enzyme concentration may have to be titrated based on cell type and density, lane1: 200U, lane2: 100U, lane3: 25U. The ‘laddering’ phenomenon seen with MNase treatment is common due to the specific cleavage of DNA by MNase between nucleosomes.

NOTE: Optional DNA Shearing Method

Micrococcal Nuclease Treatment

1. Add appropriate units of MNase based on prior optimization of MNase to effectively shear crosslinked chromatin. This can range from 25U to 200U or more for each IP performed.

2. Incubate at 37°C, 10 minutes.

3. Add 30 µL 200 mM EGTA to stop the reaction.

Procedure D: Incubate With Specific Antibody

1. If the sample (from Procedure B

Step 11

,

on page 12 ) was frozen, thaw.

2. Transfer supernatant to a 15 mL tube and add 5 volumes of IP dilution buffer containing protease inhibitors (tablet from Roche, add before use).

3. Pre-equilibriate protein A Sepharose

beads by washing 100 μL beads with 1 mL IP dilution buffer, pellet cells by centrifuging for 2 minutes at 2,000 rpm at 4°C in a microcentrifuge. Remove

~ 800 μL supernatant.

4. Pre-clear chromatin by adding 200 μL pre-equilibrated Protein A Sepharose beads.

Chapter 2 | Chromatin Immunoprecipitation Assay

14

5. Incubate on a rotating platform at 4°C for 30 minutes.

6. Centrifuge at 2,000 rpm for 2 minutes at 4°C in a swinging bucket rotor.

7. Transfer supernatant to a new 15 mL tube and discard beads.

8. Add 10 to 15 μg of antibody per IP. Usually, a negative control is performed using the same number of cells with a non-specific IgG or no antibody (mock IP) control.

NOTE: The amount of antibody to be added is dependent on quality, affinity, specificity, and type of antibody used. Users may have to titrate the amount of antibody used for each IP.

9. Incubate on rotating platform at 4°C overnight (or for at least 3 hours at RT).

DAY 2

Procedure E: Immunoprecipitate and Wash

1. Pre-equilibrate protein A Sepharose

beads by adding 1 mL IP Dilution Buffer and 200 μL beads for each IP’d sample. Centrifuge 2,000 rpm 2 minutes at 4°C.

2. Discard around 800 μL supernatant: save ~ 400 μL of beads in buffer at the bottom of the tube.

3. Transfer 400 μL beads to each sample.

4. Add PMSF to each tube sample (final concentration 1mM PMSF in final volume).

5. Incubate on rotating platform at RT for 1 to 3 hours.

6. Centrifuge at 2,000 rpm at 4°C for 4 minutes, and then discard supernatant.

7. Resuspend the pellet with 700 μL ChIP wash 1 (containing 1 mM PMSF added fresh), mix and transfer to spin-X column.

8. Incubate on rotating platform at RT for 1 minute.

9. Centrifuge at 2,000 rpm at RT for 2 minutes and discard flow-through.

10. Repeat steps 7–9.

11. Wash the beads with 700 μL ChIP wash 2 (containing 1 mM fresh PMSF).

12. Incubate on rotating platform at RT for 5 minutes.

13. Centrifuge at 2,000 rpm at RT and discard flow-through.

14. Wash the beads with 700 μL ChIP wash 3.

15. Incubate on rotating platform at RT for 5 minutes.

16. Centrifuge at 2,000 rpm at RT and discard flow-through.

17. Wash the beads with 700 μL TE (10 mM Tris-HCl pH 8, 1 mM EDTA).

18. Incubate on rotating platform at RT for 1 minute.

19. Centrifuge at 2,000 rpm at RT and discard flow-through.

20. Repeat steps 17 through 19.

21. Transfer the spin-X column with beads to a dolphin-nose tube.

22. Add 200 μL Elution Buffer to the column.

23. Incubate at 65°C for 30 minutes.

24. Centrifuge at 3,000 rpm at RT for 2 minutes.

25. Add 200 μL Elution Buffer to the column.

26. Centrifuge at 3,000 rpm at RT for 2 minutes. This 400 μL eluted sample is the “enriched” or “IP’d” sample.

Chapter 2 | Chromatin Immunoprecipitation Assay

15

Procedure F: Reverse Crosslinks

1. Add 5 μL Proteinase K (20mg/mL) per 100 μL of negative control or IP sample, mix well. (20 μL for

400 μL of eluted sample.)

2. Incubate in incubator at 65°C overnight.

DAY 3

Procedure G: Cleanup De-crosslinked Samples

1. USB PrepEase DNA Clean-Up Kit (see

Cleanup of Double-Stranded DNA

on page 23

).

NOTE: 2. IP efficiency can be checked at this stage in the protocol using polymerase chain reaction (PCR) and designing primer sets against regions that are known to be bound by the protein of interest and immunoprecipitated using the antibody being investigated. A significant increase or enrichment for the specific target should be observed for the IP condition compared to the Ab- control. Using quantitative real-time PCR, Affymetrix has routinely obtained >8-fold enrichment for IP samples compared to the Ab- samples.

Procedure H: PCR Amplify Immunoprecipitated DNA Targets

NOTE: Dilute Sequenase

stock with Sequenase Dilution Buffer (included with enzyme) to

1.3 U/µL. Four microliters of this 1.3 U/µL working stock will be needed for each sample being amplified.

1. Use 10 μL of IP’d or negative control sample for initial round of linear amplification.

2. Set up first round reaction. Set up 1 reaction for single array products (e.g., Human Promoter 1.0R

Array). Setup 3 reactions for multi-array sets (e.g., Human Tiling 2.0R Array Set).

Table 2.2

Component

Purified DNA

5X Sequenase ™ Reaction Buffer *

Primer A (200 µM) †

Total Volume

* Included with enzyme.

† Primer A: GTTTCCCAGTCACGGTC(N)

9

(HPLC purified)

Volume for 1 Rxn

10 µL

4 µL

4 µL

18 µL

3. Cycle conditions: Random priming.

A.

B.

C.

D.

95°C for 4 minutes.

Snap cool samples on ice.

10°C hold.

Prepare first cocktail ( Table 2.3

).

Chapter 2 | Chromatin Immunoprecipitation Assay

16

Table 2.3 First Cocktail

Component

20 mg/mL BSA

0.1 M DTT

25 mM dNTPs

Diluted Sequenase ™ (1/10 from 13 U/µL stock)

Total Volume

Volume for 1 Rxn

0.1 µL

1 µL

0.5 µL

1 µL

2.6 µL

E.

Add 2.6 μL per sample.

F.

Mix well by pipetting, and put the sample back in thermocycler block.

G.

10°C for 5 minutes.

H.

R.

Ramp from 10°C to 37°C over 9 minutes.

I.

Q.

37°C for 8 minutes.

J.

P.

95°C for 4 minutes.

K.

O.

Snap cool on ice.

L.

N.

10°C hold.

M.

Add 1.0 μL of 1.3U/μL Sequenase ™ to each sample.

10°C for 5 minutes.

Ramp from 10°C to 37°C over 9 minutes.

37°C for 8 minutes.

Repeat from J) to P) for 2 more cycles.

4°C hold.

4. For each IP, purify with Microspin S-300 HR (GE Healthcare) columns (2 columns per reaction) as follows:

A.

B.

C.

D.

E.

F.

G.

Add 20 μL of 10 mM TE pH 8.0 to each reaction.

Spin 2 columns (A & B) at 3,000 rpm for 1 minute, discard flow-through.

Transfer reaction volume (~ 43 μL) to column A, while equilibrating column B with 300 μL of

10 mM Tris pH 8.0.

Spin both columns at 3,000 rpm for 1 minute, keep flow-through from column A (sample) and discard flow-through of column B (Tris buffer).

Transfer flow-through of column A to column B with new collection tube.

Spin at 3,000 rpm for 2 minutes.

Collect ~ 56 μL of first round purified DNA per reaction.

5. Prepare dNTP/dUTP mix.

Chapter 2 | Chromatin Immunoprecipitation Assay

17

Prior to proceeding with the PCR amplification of immunoprecipitated DNA targets, prepare a dNTP mixture containing dUTP at the concentrations indicated below. Please note that this dNTP + dUTP mixture is only required for the PCR amplification reaction outlined in

Table 2.4

and not in the Sequenase ™ reaction setup in

Table 2.3

.

dCTP – 10 mM dATP – 10 mM dGTP – 10 mM dTTP – 8 mM dUTP – 2 mM

Store at –20°C.

6. PCR Mix Setup:

Table 2.4

Component

First-round DNA from Step 4

10X PCR Buffer

25 mM MgCl2 *

10 mM dNTPs + dUTP

100 µM Primer B †

5 U/µL Taq Polymerase

Nuclease-free Water

Total Volume

* Add MgCl

2

if using magnesium-free 10X PCR Buffer.

† Primer B (GTTTCCCAGTCACGGTC)

Volume for 1 Rxn

20 µL

10 µL

3 µL

3.75 µL

4 µL

2 µL

57.25 µL

100 µL

7. Cycle conditions:

A.

15 cycles

1

1) 95°C 30 seconds.

2) 45°C 30 seconds.

3) 55°C 30 seconds.

4) 72°C 1 minute.

15 cycles 1

B.

1) 95°C 30 seconds.

2) 45°C 30 seconds.

3) 55°C 30 seconds.

C.

4) 72°C 1 minute.

For every subsequent cycle add 5 seconds.

E.g., cycle 1: 60 seconds, cycle 2: 65 seconds, etc...

4°C hold.

8. Check amplified DNA on 1% agarose gel.

1 Number of PCR amplification cycles may require optimization. QPCR can be used to evaluate enrichment of immunoprecipitated sample.

Figure 2.2 PCR-amplified ChIP targets from HL-

60 cells immunoprecipitated with an Sp1 antibody. Replicate PCR reactions (lanes 1 to 3) were performed on the same IP sample and product sizes ranged from 200 bp to over 2 Kb but the actual product sizes may vary depending on original size of sheared chromatin.

Chapter 2 | Chromatin Immunoprecipitation Assay

18

9. Purify PCR samples using USB PrepEase DNA Clean-Up kit (see

Cleanup of Double-Stranded DNA

on page 23 ).

10. Measure DNA using a NanoDrop or other UV-vis spectrophotometer. Normally, greater than 9 μg of amplified DNA is obtained from each reaction.

NOTE: Maintenance of IP enrichment post-amplification is crucial in obtaining good array results. QPCR should be performed to post-amplified samples to ensure that differences between the IP and Ab- samples are maintained. Primer sets can be designed for DNA regions that are known to be specifically immunoprecipitated using the antibody of interest.

Chapter 2 | Chromatin Immunoprecipitation Assay

19

Procedure I: Fragment Amplified Targets

1. Fragment the samples using the appropriate table below depending on what array type the target will be hybridized to.

Table 2.5 Fragmentation Mix for single arrays (e.g., Human Promoter 1.0R Array)

Fragmentation of ds cDNA

Component Volume/Amount in 1 Rxn

ds cDNA

Nuclease-free Water

10X APE 1 Reaction Buffer

Uracil-DNA Glycosylase (UDG) (2U/ µL)

Human Apurinic/Apyrimidinic Endonuclease 1

(APE 1) (10 U/µL)

Total Volume

7.5 µg up to 32.2 µL

4.8 µL

4.0 µL

7.0 µL

48.0 µL

Table 2.6 Fragmentation Mix for multi-array sets (e.g., Human Tiling 2.0R Array Set)

Fragmentation of ds cDNA

Component Volume/Amount in 1 Rxn

ds cDNA

Nuclease-free Water

10X APE 1 Reaction Buffer

Uracil-DNA Glycosylase (UDG) (2U/ µL)

Human Apurinic/Apyrimidinic Endonuclease 1

(APE 1) (10 U/µL)

Total Volume

9.0 µg up to 32.2 µL

4.8 µL

4.0 µL

7.0 µL

48.0 µL

2. Set up fragmentation mix according to either

Table 2.5

or

Table 2.6

) Flick-mix and spin down the

tubes.

3. Incubate the reactions at:

37°C for 1 hour.

93°C for 2 minutes.

4°C for at least 2 minutes.

4. Flick-mix, spin down the tubes, and transfer 45 μL of the sample to a new tube.

5. The remainder of the sample is to be used for fragmentation analysis using a Bioanalyzer or agarose gel. Please see the Reagent Kit Guide that comes with the RNA 6000 LabChip Kit for instructions.

If not labeling the samples immediately, store the fragmented DNA at –20°C.

Chapter 2 | Chromatin Immunoprecipitation Assay

20

Figure 2.3 Bioanalyzer trace of fragmentation products following treatment of amplified ChIP targets with UDG and APE 1. Independently amplified Sp1 IP or Ab- samples from HL-60 cells were fragmented according to the protocol and products were analyzed on an Agilent

Bioanalyzer with the RNA 6000 Nano LabChip Kit. Analyzing fragmented

DNA on the RNA 6000 LabChip is recommended because it quickly assesses the degree and uniformity of the fragmented products.

Procedure J: Label Fragmented Double-Stranded DNA

1. Prepare the Double-Stranded DNA Labeling Mix as described in

Table 2.7

.

Table 2.7 Double-Stranded DNA Labeling Mix

Component

5x TdT Reaction Buffer

Terminal Deoxynucleotidyl Transferase (rTdT),

Recombinant, (30 U/uL)

DNA Labeling Reagent, DLR, 10 mM

Total Volume

Volume in 1 Rxn

12 µL

2 µL

1 µL

15 µL

2. Add 15 μL of the Double-Stranded DNA Labeling Mix to the DNA samples, flick-mix, and spin them down.

3. Incubate the reactions at:

37°C for 60 minutes.

70°C for 10 minutes.

4°C for at least 2 minutes.

4. Remove 2 μL of each sample for gel-shift analysis (refer to the GeneChip

® Whole Transcript (WT)

Sense Target Labeling Assay Manual).

3

Hybridization and Array Processing

Procedure A: Hybridize Labeled Target on the Arrays

This Procedure requires the use of the GeneChip

®

Hybridization, Wash, and Stain Kit (P/N 900720).

1. Prepare the Hybridization Cocktail in a 1.5 mL RNase-free microfuge tube as shown in

Table 3.1

and

Table 3.2

, below depending on what array type the target will be hybridized to.

Table 3.1 Hybridization Cocktail for single tiling arrays (e.g., GeneChip ® Human Promoter 1.0R Array)

Component Volume in 1

Rxn

Final Concentration or Amount

Fragmented and Labeled DNA Target ~ 60.0 µL * ~ 7.5 µg

Control Oligonucleotide B2

2X Hybridization Mix †

DMSO

Nuclease-free Water

Total Volume

3.3 µL

100 µL

14.0 µL up to 200.0 µL

200.0 µL

50 pM

1X

7%

* This volume is 56 µL if a portion of the sample was set aside for gel-shift analysis.

† Available in the GeneChip ® Hybridization, Wash, and Stain Kit.

Table 3.2 Hybridization Cocktail for use with serial hybridizations (e.g., GeneChip ®

Human Tiling 2.0R Array Set and GeneChip ® Mouse Tiling 2.0R Array Set)

Component Volume in 1

Rxn

Final Concentration or Amount

Fragmented and Labeled DNA Target

Control Oligonucleotide B2

2X Hybridization Mix †

DMSO

Nuclease-free Water

Total Volume

~ 60.0 µL *

4 µL

120 µL

16.8 µL up to 240.0 µL

240.0 µL

~ 9.0 µg

50 pM

1X

7%

*

This volume is 58 µL if a portion of the sample was set aside for gel-shift analysis.

† Available in the GeneChip ® Hybridization, Wash, and Stain Kit.

2. Flick-mix, and centrifuge the tube.

3. Heat the Hybridization Cocktail at 99°C for 5 minutes. Cool to 45°C for 5 minutes, and centrifuge at maximum speed for 1 minute.

4. Inject ~ 200 μL of the specific sample into the array through one of the septa (see

Figure 3.1

for

location of the septa on the array). Save the remaining hybridization cocktail in –20°C for future use.

5. Place array in 45°C hybridization oven, at 60 rpm, and incubate for 16 hours.

6. After hybridization, remove the hybridization cocktail for future use.

Chapter 3 | Hybridization and Array Processing

22

Figure 3.1 GeneChip ® Probe Array

Plastic cartridge

Front

Probe array on glass substrate

Notch

Septa

Back

Procedure B: Array Wash, Stain and Scan

For instructions on array washing, staining and scanning please refer to the GeneChip

®

Expression Wash,

Stain and Scan User Manual (P/N 702731).

A

Cleanup of Double-Stranded DNA

Cleanup of Double-Stranded DNA

This Step requires the use of the PrepEase

®

DNA Clean-Up Kit (PrepEase

®

DNA Clean-Up Kit, P/Ns

78758, 78759).

Brief Protocol for Concentration, Desalination and/or Removal of Enzymes

IMPORTANT: Check that ethanol was added to NT3 Buffer before starting.

1. Adjust DNA binding conditions

A.

B.

Add 5 volumes of N2P Buffer to 1 volume of sample (e.g., 500 μL N2P Buffer and 100 μL sample).

Mix well.

2. Bind DNA sample to column

A.

B.

Place PrepEase ® Clean-Up Column into a 2 mL PrepEase ® Collecting Tube.

Pipet the sample directly into the center of the column.

C.

D.

Centrifuge 1 min at 11,000 x g.

Discard flow-through.

3. Wash column

A.

B.

Add 600 μL NT3 Buffer to column.

Centrifuge 1 min at 11,000 x g.

Discard flow-through. Place column back into collecting tube.

C.

4. Dry column

Centrifuge 2 min at 11,000 x g.

5. Elute DNA

A.

B.

C.

D.

Place the column into a clean 1.5 ml microcentrifuge tube.

Add 15-50 μL NE Buffer to column.

Incubate at room temperature for 1 min.

Centrifuge 1 min at 11,000 x g.

Brief Protocol for PCR Purification

IMPORTANT: Check that ethanol was added to NT3 Buffer before starting.

1. Adjust DNA binding conditions

A.

B.

Add 5 volumes of N2P Buffer to 1 volume of sample (e.g., 250 μL N2P Buffer and 50 μL sample).

Mix well.

2. Continue with

Step 2 to Step 5

of the

Brief Protocol for Concentration, Desalination and/or Removal

of Enzymes on page 23

.

Appendix A | Cleanup of Double-Stranded DNA

24

Brief Protocol for DNA Purification from Chromatin Immunoprecipitation (ChIP) Assay

IMPORTANT: Check that ethanol was added to NT3 Buffer before starting.

1. Adjust DNA binding conditions

A.

B.

Add 5 volumes of N2P Buffer to 1 volume of sample (e.g. 1000 μL N2P Buffer and 200 μL sample).

Mix well.

2. Bind DNA sample to column

A.

B.

Place PrepEase ® Clean-Up Column into a 2 ml PrepEase ® Collecting Tube.

Pipet 700 μL of the sample directly into the center of the column.

C.

D.

Centrifuge 1 min at 11,000 x g.

Discard flow-through.

E.

Repeat

Step B

3. Wash column

to Step D for the remaining sample.

A.

B.

Add 600 μL NT3 Buffer to column.

Centrifuge 1 min at 11,000 x g.

Discard flow-through. Place column back into collecting tube.

C.

4. Dry column

Centrifuge 2 min at 11,000 x g.

5. Elute DNA

A.

B.

C.

Place the column into a clean 1.5 ml microcentrifuge tube.

Add 30-40 μL NE Buffer to column.

Incubate at room temperature for 1 min.

Centrifuge 1 min at 11,000 x g.

D.

6. Take 2 μL from each sample to determine the yield by spectrophotometric UV measurement at 260 nm, 280 nm and 320 nm:

Concentration of Double-Stranded cDNA (μg/μL) =

[A

260

- A

320

] x 0.05 x dilution factor

μg DNA = eluate in μL x DNA in μg/μL

Contact Information

Affymetrix, Inc.

3420 Central Expressway

Santa Clara, CA 95051

USA

Email: [email protected]

Tel: 1-888-362-2447 (1-888-DNA-CHIP)

Fax: 1-408-731-5441

Affymetrix UK Ltd

Voyager, Mercury Park,

Wycombe Lane, Wooburn Green,

High Wycombe HP10 0HH

United Kingdom

Email: [email protected]

UK and Others Tel: +44 (0) 1628 552550

France Tel: 0800919505

Germany Tel: 01803001334

Fax: +44 (0) 1628 552585

Affymetrix Japan, K. K.

ORIX Hamamatsucho Bldg, 7F

1-24-8 Hamamatsucho, Minato-ku

Tokyo 105-0013

Japan

Email: [email protected]

Tel: +81-3-6430-4020

Fax: +81-3-6430-4021

USB Corporation

26111 Miles Road

Cleveland, Ohio 44128 USA www.usbweb.com

B

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