Whole genome amplification protocol for ChIP-chip

Introduction

The technique of chromatin immunoprecipitation (ChIP) has proven to be a powerful tool, allowing the detection of protein-DNA interactions in living cells. Hybridization of ChIP samples to DNA microarrays (i.e. the ChIP-chip assay) allows a global analysis of binding sites for transcription factors and components of the transcriptional machinery, as well as of chromatin modification patterns. However, a single ChIP sample does not yield enough DNA for hybridization to a genomic tiling array. Therefore, we have adapted the standard protocol for whole genome amplification using the Sigma GenomePlex WGA kit to amplify our ChIP sample (O'Geen et al., 2006). Using Oct4 ChIP-chip assays as an example, we have compared the quality of ChIP-chip data derived from 1) WGA amplified ChIP samples, 2) a pool of 10 ChIP samples without further amplification, and 3) linker-mediated PCR (LMPCR) amplification of ChIP samples (Figure 1). Based on the low background, reproducibility, and the fact that a single WGA amplified ChIP sample can provide sufficient material for several array hybridizations, we recommend the WGA protocol for ChIP-chip analyses. We have successfully tested our new ChIP amplification protocol on a variety of different factors (E2F family members, KAP1, CtBP2, ZNF217) as well as on histone modifications (H3me3K9, H3me3K27, H3me3K4) (Krig et al., 2007; O'Geen et al., 2007). Another benefit of the WGA amplification method is the ability to perform a second round of amplification from the initial WGA product if a higher DNA yield is required. We have applied the re-amplification protocol to KAP1 amplicons that were hybridized to a whole genome tiling array set consisting of 38 arrays (O'Geen et al., 2007). Detailed protocols for ChIP assays from mammalian cells and tissue samples, as well as preparation of amplicons can be found on the Farnham Lab website.

Procedure

The Whole Genome Amplification using Sigma GenomePlex Kit was adapted from the manufacturer's protocol. Since the input material (ChIP sample or total chromatin) is sonicated chromatin, the initial series of fragmentation steps can be skipped - proceed straight to library preparation.

Library Preparation

1. For the total chromatin sample, measure the concentration of reverse crosslinked, QIAquick purified DNA and add 10ng to a total volume of 10µl with H₂ O. For the ChIP sample, the concentration of nucleic acid is usually too low to get accurate quantitation. Typically the entire 50µl of reverse crosslinked, QIAquick purified DNA is lyophilized and resuspended in 10µl of H₂ 0 (comment 1 and comment 2);
2. Add 2µl 1X Library Preparation Buffer to 10µl of input material;
3. Transfer samples to strip tubes or individual thin walled 0.2ml PCR tubes;
4. Add 1µl Library Stabilization Solution, vortex or mix by pipetting. Quick spin and place at 95°C for 2 minutes in thermal cycler;
5. Immediately cool on ice, quick spin again;
6. Add 1µl Library Preparation Enzyme, vortex or mix by pipetting and quick spin if necessary;
7. Incubate in thermal cycler as follows:
   16°C for 20' (cycler should be precooled to this temperature)
   24°C for 20'
   37°C for 20'
   75°C for 5'
   4°C hold;
8. Quick spin if necessary and either proceed to first amplification or freeze at -20°C for up to three days.

Amplification (round 1)

1. Prepare master mix for each sample containing:
   7.5µl of 10X Amplification Master Mix
   47.5µl Nuclease-free H₂ 0
   5µl WGA DNA polymerase
   (For multiple samples, multiply above volumes by the number of samples then add 1/10 volume extra of each component);
2. Add 60µl master mix to each sample, vortex or mix by pipetting and quick spin if necessary;
3. Incubate in thermal cycler as follows:
   95°C for 3', then 14 cycles of
   94°C for 15"
   65°C for 5', then
   4°C hold
   At this point, amplified material is stable and can be stored at -20°C;
4. Purify samples using QIAquick PCR cleanup columns or analogous product. (Since the amplified material contains both single- and double-stranded DNA that can be effectively labeled, the column purification method used should recover both.) (comment 3).

At this stage, the purification column eluates for total and immunoprecipitated samples should be readily quantifiable by nanodrop, spectrometer, or dye intercalation, e.g. picogreen (dye intercalation may underestimate amount due to single strand product). Optimally, total recovery for immunoprecipitated samples will be in the 1-4µg range. This gives enough material for several labelings for downstream microarray analysis. If yields are less, or more product is desired, re-amplify material using Sigma GenomePlex WGA Reamplification Kit. However, if less than 1µg is obtained, it is likely that a problem occurred in the initial steps-do not use the sample for reamplifcation (comment 4 and comment 5).

Reamplification (round 2)

1. Add 20ng purified amplification product in 10µl volume to strip tubes or individual thin walled 0.2ml PCR tubes. (Always generate round 2 from both round 1 of the ChIP sample and round 1 of the total chromatin to ensure that both samples undergo the same manipulations);
2. Prepare master mix for each sample containing:
   7.5µl of 10X Amplification Master Mix
   47.5µl Nuclease-free H₂ 0
   5µl WGA DNA polymerase
   (For multiple samples, multiply above volumes by the number of samples then add 1/10 volume extra of each component);
3. Add 60µl master mix to each sample, vortex or mix by pipetting and quick spin if necessary;
4. Incubate in thermal cycler as follows:
   95°C for 3' , then 14 cycles of
   94°C for 15"
   65°C for 5', then
   4°C hold
   At this point, amplified material is stable and can be stored at -20°C;
5. Purify samples using QIAquick PCR cleanup columns or analogous product. (Since the amplified material contains both single- and double-stranded DNA that can be effectively labeled, the column purification method used should recover both.)

Materials & Reagents

• ChIP sample, total chromatin
• WGA1 Sigma GenomePlex® Whole Genome Amplification (WGA) Kit
• WGA3 Sigma GenomePlex® WGA Reamplification Kit (comment 6)

Reviewer Comments

Reviewed by: Angelika Mitterweger, Becker lab, Adolf-Butenandt-Institut, München, Germany

1. I keep 3µl from the reversed cross-linked material for a control q-PCR analysis, which can serve as a quality control or independent confirmation of the result.
2. I transfer the remaining 47µl of reverse crosslinked material into a PCR tube. Lyophylization in the PCR tube (which is placed into a normal 1.5ml reaction tube for support) minimizes losses. I also try to avoid complete lyophylization since losses may occur upon resuspension. Rather I dry the sample down to below 10µl and adjust the volume to 10µl.
3. We use the GFX-kit from Amersham with good and consistent results.
4. At this stage you may want to check IPs and amplification products for linearity of amplification by q-PCR.
5. The OD readings of the amplification reaction (after round 1) provide a quality control. 260/280 ratios should be above 1.7, 260/230 ratios above 1.6. Lower values point to problems and correlate with inefficient (re-) amplification.
6. Sigma also provides a WGA2 kit, which combines the WGA1 and WGA3 kits at a better price.

Figures

Figure 1: Comparison of three different sample preparation methods for Oct4 ChIP-chip assays in Ntera2 cells. The hybridization profile of a 300 kb region of chromosome 8 surrounding the EXT1 gene is shown for samples prepared by LMPCR amplification of ChIP samples (top panel), pooling 10 ChIP samples without further amplification (middle panel), or WGA amplification of ChIP samples (bottom panel). Oct4 binding sites confirmed by PCR are indicated with arrows.

References

1. Krig, S.R., Jin, V.X., Bieda, M., O'Geen, H., Green, R., and Farnham, P.J. (2007) Identification of genes directly regulated by the oncogene ZNF217 using chromatin immunoprecipitation (ChIP)-chip assays, J Biol Chem , 282(13) : 9703-12.
2. O'Geen, H., Nicolet, C.M., Blahnik, K., Green, R., and Farnham, P.J. (2006) Comparison of sample preparation methods for ChIP-chip assays, Biotechniques , 41(5) : 577-580.
3. O'Geen, H., Squazzo, S.L., Iyengar, S., Blahnik, K., Rinn, J.L., Chang, H.Y, Green, R. and Farnham, P.J. (2007) A genome-wide analysis of KAP1 binding suggests a wide-spread auto-regulation of KRAB-ZNF genes, PloS Genetics , in press.