The T7-based IVT protocol for linear amplification of genomic DNA is shown in Figure 1. Since ChIP DNA is fragmented randomly and lacks a universal sequence for priming, we optimized a terminal transferase step to add polyT tails to the 3' ends of each DNA strand. We are able to generate tails with a tight distribution around an optimal length of 30 bps by including a low concentration of a terminating dideoxycytidine nucleotide (data not shown). An anchored primer adapter was designed that contains a 5' T7 promoter and a 3' polyA (18 bps in length) stretch terminating in a single C, G, or T base. This adapter is annealed to the genomic DNA fragments, and Klenow is added for second strand synthesis. In addition to extending the primer adapter, the 3'-5' exonuclease activity of Klenow should trim 3' polyTs in excess of the 18 bps complementary to the adapter, and allow fill in of the appropriate complementary bases (Fig 1). For each dsDNA species present in the starting material, these reactions generate two dsDNAs with T7 promoters at opposite ends.

The reaction products are used as template for IVT using the Eberwine method 14. From a starting material of ChIP DNA from ~2 × 10⁸ yeast cells this protocol yields between 30 and 60 μg RNA. This yield compares favorably with R-PCR which yields up to 15 μg DNA from equivalent starting material. To more precisely evaluate the efficiency and fidelity of the linear amplification protocol, we generated a large pool of starting material by restricting yeast genomic DNA with Alu I (cuts AGCT, leaving blunt ends) and gel-purifying fragments within a size range of 100–700 bps. We subjected 50 ng of the Alu I digest to amplification by IVT and by R-PCR. The IVT yielded almost 80 μg RNA, while the R-PCR yielded 12 μg DNA (Table 2). We compared the Alu I digest with the amplified products by gel-electrophoresis (Fig 2). The IVT product retains essentially the same size distribution as the starting material. In contrast, the R-PCR product appears to under-represent lower molecular weight species.

While analyzing the size distribution of the IVT product we noted a low molecular weight species that roughly corresponds in size to the primer adapter. IVT protocols are reportedly sensitive to excess starting concentrations of the T7 primer. Baugh and colleagues were able to improve yields when amplifying mRNA by decreasing the concentration of T7 primer 16. When we limited the mass of T7 primer adapter to approximately five times that of the starting material with a lower limit of 50 ng (by decreasing the second strand synthesis reaction volume), this low molecular weight species disappeared, without decreasing overall yields (data not shown). In this way, we were able to efficiently amplify just 2.5 ng from the Alu I digest with a yield of 10.3 μg (Table 2).

We went on to evaluate the fidelity of the linear amplification protocol by microarray analysis. Restriction by Rsa I (cuts GTAC, leaving blunt ends) and gel-purification were used to generate a second population of DNA. Since Alu I and Rsa I cut at different sites, the resulting digests contain different distributions of DNA species. 50 ng of each pool were amplified by IVT. DNA probes, generated by reverse transcribing the amplified RNA, were fluorescently labeled (Cy5 for Alu I, Cy3 for Rsa I), pooled, and hybridized to microarrays containing the yeast open reading frames. After hybridization, the microarrays were washed and scanned, and Cy5/Cy3 ratios calculated. We also hybridized Alu I and Rsa I probes generated by R-PCR from 50 ng starting material, as well as probes generated by direct labeling of 1 μg starting material.

First, we assessed the reproducibility of the IVT protocol. Three Alu I / Rsa I datasets were independently generated using the IVT protocol. Each dataset contains 4,481 ratios that each reflect the relative abundance of DNA corresponding to a specific array feature (yeast ORF) in the two digests. We found the IVT protocol to be highly reproducible: correlation coefficients calculated between the replicate datasets averaged 0.98. This reproducibility is maintained even when only 5 ng starting material are used (correlation of 0.97). The direct labeling and R-PCR protocols are also highly reproducible, with mean correlations of 0.93 and 0.91, respectively (Fig 3A).

Next, we assessed the fidelity of amplification by comparing the IVT and R-PCR datasets with the direct labeling dataset. We assumed the direct labeling dataset to most accurately represent the true distribution of species in the Alu I and Rsa I digests, since no amplification was required to obtain these data. The three datasets collected for each protocol were averaged and correlation coefficients calculated between the resulting composite datasets. The correlation between the IVT dataset and the direct labeling dataset is 0.96 (Fig 3A). This value increases to 0.98 when features with ratios close to 1 are excluded from the calculation. The correlation between the R-PCR dataset and the direct labeling dataset is 0.68 (Fig 3A). It rises to 0.80 when features with ratios close to 1 are excluded. Correspondingly, the highest ranking features in the direct labeling dataset overlap extensively with the highest ranking features in the IVT dataset and, to a lesser extent, with the highest ranking features in the R-PCR dataset (Fig 3B).

The high correlation between the IVT and direct labeling datasets is evident in the scatter plot shown in Fig 4. An analogous plot portrays the lower correlation between the R-PCR and direct labeling datasets. These plots also illustrate another difference between the amplification protocols: an examination of best fit lines calculated for each of the plots suggests that dynamic range is increased by IVT amplification (slope > 1), but decreased by R-PCR amplification (slope < 1). To quantify dynamic range, we determined the ratio values for data points two standard deviations above and below the mean, and calculated the range between these values by dividing the high ratio by the low ratio. For the direct labeling dataset, the high and low ratios are 3.1 and 0.31, respectively, for a range of 9.8. The range for the IVT dataset is 17.4, while that for the R-PCR dataset is 5.8 (Table 2).

Hierarchical clustering, shown in Fig 5, was used as an additional means to identify differences between the amplification protocols 20. Replicates for a given method cluster closely, confirming the high reproducibility observed for each method. Consistent with the correlation analysis, the individual replicates for the IVT and direct labeling protocol cluster together, while the R-PCR replicates cluster in a separate arm of the dendrogram. Most of the 4,481 features shown in the cluster diagram exhibit consistent coloring across the diagram, indicating that the direction of enrichment is consistent for all replicates. However, the cluster diagram highlights two groups of genes (purple stripes) whose ratios in the R-PCR dataset are inconsistent with their ratios in the direct labeling and IVT datasets.

Genomic DNA used in amplification protocols was obtained either by ChIP or by restriction digests. ChIP DNA was fragmented by sonication and isolated using antibody against di-methyl-H3 K4 as described previously 6. Restricted genomic DNA was prepared as follows: yeast genomic DNA isolated by bead lysis, phenol/chloroform extraction, and ethanol precipitation, was restricted either with Alu I or with Rsa I (New England BioLabs (NEB)). Digested products underwent electrophoresis on a 2% agarose gel. Restriction fragments in the 100–700 bp size range were excised from the gel and purified using the QIAquick Gel Extraction Kit (Qiagen).

Calf intestinal alkaline phosphatase (CIP) (NEB) was used to remove 3' phosphate groups from DNA samples prior to IVT. Up to 500 ng DNA were incubated with 2.5 U enzyme in a 10 μL volume with the supplied buffer at 37°C for 1 hour. The reaction was cleaned up with the MinElute Reaction Cleanup Kit (Qiagen) per manufacturer instructions except that the elution volume was increased to 20 μL.

PolyT tails were generated using terminal transferase (TdT) as follows. Up to 50 ng of CIP-treated template DNA were incubated for 20 minutes at 37°C in a 10 μL solution containing 20 U TdT (NEB), 0.2 M potassium cacodylate, 25 mM Tris-HCl pH 6.6, 0.25 mg/ml BSA, 0.75 mM CoCl₂, 4.6 μM dTTP and 0.4 μM ddCTP. The reaction was halted by the addition of 2 μL of 0.5 M EDTA pH 8.0, and product isolated with the MinElute Reaction Cleanup Kit (Qiagen), increasing the elution volume to 20 μL.

Second strand synthesis and incorporation of the T7 promoter sequence was carried out as follows: the 20 μL tailing reaction product was mixed with 0.6 μL of 25 μM T7-A₁₈B primer (5'-GCATTAGCGGCCGCGAAATTAATACGACTCACTATAGGGAG(A)₁₈ [B], where B refers to C, G or T), 5 μL 10X EcoPol buffer (100 mM Tris-HCl pH 7.5, 50 mM MgCl₂, 75 mM dTT), 2 μL 5.0 mM dNTPs, and 20.4 μL nuclease-free water. In experiments with 10–50 ng starting material, the end primer concentration was kept at 300 nM, while the reaction volume was scaled down to maintain an end concentration of 1 ng/ul starting material. For starting amounts less than 10 ng, the volume was kept at 10 μL. If necessary, volume reduction of the eluate from the TdT tailing was performed in a vacuum centrifuge on medium heat. Samples were incubated at 94°C for 2 minutes to denature, ramped down at -1 C°/sec to 35°C, held at 35°C for 2 minutes to anneal, ramped down at -0.5 C°/sec to 25°C and held while Klenow enzyme was added (NEB) to an end concentration of 0.2 U/μL. The sample was then incubated at 37°C for 90 minutes for extension. The reaction was halted by addition of 5 μL 0.5 M EDTA pH 8.0 and product isolated with the MinElute Reaction Cleanup Kit (Qiagen), increasing the elution volume to 20 μL.

Prior to in vitro transcription, samples were concentrated in a vacuum centrifuge at medium heat to 8 μL volume. The in vitro transcription was performed with the T7 Megascript Kit (Ambion) per manufacturer's instructions, except that the 37°C incubation was increased to 16 hours. The samples were purified with the RNeasy Mini Kit (Qiagen) per manufacturer's RNA cleanup protocol, except with an additional 500 μL wash with buffer RPE. RNA was quantified by absorbance at 260 nm, and visualized on a denaturing 1.25X MOPS-EDTA-Sodium Acetate gel.

For each sample to be analyzed by microarray, 4 μg amplified RNA were primed with 5 μg random hexamers and 5 μg oligo dT and reverse-transcribed, incorporating aa-dUTP, as described at http://www.microarrays.org. Note: the presence/absence of the oligo dT primer does not affect the RT and labeling efficiency. RT was halted with 10 μL 0.5 M EDTA, and RNA hydrolyzed by the addition of 10 μL 1 N NaOH and incubation at 65°C for 15 minutes. Reaction cleanup and labeling with monofunctional reactive Cy5 (Alu I digest) or Cy3 (Rsa I digest) dye was carried out as described in 22 and at http://www.microarrays.org.

R-PCR samples were generated from 50 ng restricted DNA using the Round A/ Round B protocol described in 912. Briefly, Sequenase (Amersham) was used for two cycles of synthesis using a degenerate primer (5'-GTTTCCCAGTCACGATCNNNNNNNNN-3' ('round A'). The resulting end-tagged DNAs were subjected to 30 cycles of PCR using the following primer: 5'-GTTTCCCAGTCACGATC-3'. Amino-allyl dUTP was incorporated during the PCR step as described in 22, and at http://www.microarrays.org. Amplified DNA was purified with a QIAquick PCR purification kit (Qiagen). For microarray analysis, 7 μg of each probe were fluorescently labeled by incubation with monofunctional reactive Cy5 (Alu I digest) or Cy3 (Rsa I digest) dye as described 22.

Unamplified DNA samples for labeling were generated from 1 μg restricted DNA using Klenow fragment and random primers, and incorporating amino-allyl dUTP, as described at http://www.microarrays.org.

Yeast ORFs were amplified from a set of 6,218 plasmids using universal primers as described 1. This set of ORFs was printed on separate slides, hydrated, and snap-dried as described 1. Mixed Cy5/Cy3-labeled probes were hybridized to microarrays for 12–15 h at 60°C. After hybridization, microarrays were washed as described 1 and scanned with a GenePix 4000B scanner with GENEPIX PRO 4.0 software (Axon Instruments). Microarray data are available at http://www.schreiber.chem.harvard.edu

The Cy5/Cy3 ratio for each array element was calculated using GENEPIX PRO 4.0, log₂ transformed, and normalized using the default computed normalization method used by the Stanford Microarray Database 23. We applied a high stringency data selection, keeping only microarray elements for which at least 80% of the measurements within a set of experiments had fluorescence intensity in both channels at least 3-fold over background intensity. 4,481 array elements passed these selection criteria.

IVT, R-PCR, and direct labeling procedures and hybridizations were each carried out in triplicate. To assess reproducibility of each method, the correlation coefficients were calculated for all possible combinations of pairs within each set of replicates, and then averaged. Next, a single averaged dataset was generated for each method by determining the geometric mean for each array element from a given set of replicates. To assess the degree of similarity among the different methods, correlation coefficients were calculated in pair-wise comparisons of these averaged datasets. An additional correlation coefficient was calculated between averaged datasets using only those features whose ratios in the direct labeling dataset reflect at least a 1.5-fold change, either up or down.

Hierarchical clustering of the non-averaged replicate datasets was carried out using the Cluster program, with average linkage using Pearson correlation as the similarity metric 20. Clusters were visualized using TreeView 20. To assess the degree of overlap among the different methods, the number of features among the top 500 log₂ Cy5/Cy3 ratios was determined for each pair-wise comparison and plotted in Venn Diagrams, with the overlap region scaled to the number of features. To assess the dynamic range characteristics of the three methods, the range of the data for each method was taken as an interval extending 2 standard deviations on either side of the mean across the entire array.