enriched regions do not overlap with current JGI gene annotation within 1 kb (40%; 4,116 out of 10,179). An experimental approach to the design of a promoter microarray, based on H3K4me3-enriched regions, is therefore highly favored. Although many promoters featured on this microarray design are expected to be active in multiple tissues and stages of development, it should be noted that most comprehensive results will be obtained with this design when analyzing promoter binding events in gastrula stage embryos. The biological variation of the H3K4me3 modification is quite low in Xenopus gastrula embryos. We also performed TBP ChIP- chip and find TBP peaks overlapping with H3K4me3 peaks, as expected for active promoters. The correlation between H3K4me3 and TBP, though highly significant, is not as strong as that observed between biological replicates of H3K4me3 enrichment. However, it should be noted that TBP paralogs have been identified that are required for transcription initiation during embryogenesis [19,20,21] and some promoters decorated with H3K4me3 do not recruit TBP. It will be worthwhile to study the dynamics of TBP related factors during early Xenopus development. This work describes microarray designs which are made available to the community, thereby facilitating future studies in Xenopus embryogenesis using ChIP-chip. For the analysis of promoter binding the promoter microarray design for X. tropicalis can be used. For a genome-wide analysis of DNA binding or epigenetic regulation the five microarray set can be used. These types of genomic studies will enhance our understanding of transcriptional networks and developmental pathways and identify novel targets genes important for vertebrate development. Materials and Methods Chromatin Immunoprecipitation Animal work has been conducted according to relevant national and international guidelines and following approval of the institutional review board for animal experimentation (Dierexper- imenten commissie). Xenopus tropicalis embryos were obtained from a natural mating procedure after human chorionic gonadotropin injection, dejellied in 2% cysteine and collected at the indicated stage. Chromatin harvesting from 300 gastrula X. tropicalis embryos and ChIP (15 embryo equivalents) was performed as described [20] with two minor modifications: 12.5 ml of Prot A/G beads (Santa Cruz) were used and during reversal of the crosslinking proteinase K was omitted from the buffer. We used a-H3K4me3 (Abcam), a- H3K9ac (Upstate) and a-TBP (SL33) antibodies. RNA Isolation and cDNA Synthesis Total RNA was isolated from 20 gastrula X. tropicalis embryos using TRIzol reagent (Invitrogen). 5 mg of RNA was subjected to a reverse transcription reaction in the presence (cDNA) or absence (RT-) of SuperScriptIII (Invitrogen). Quantitative PCR 5 ml of ChIP material (0.375 embryo equivalents) or 10 times diluted cDNA or RT- was used for quantitative PCR. PCR reactions were performed on a MyIQ single color real-time PCR detection system (BioRad) using iQ SYBR Green Supermix (BioRad). Primer sequences are available in Table S3. T7 Amplification T7 amplification procedure was essentially identical as de- scribed by [10]. In short, after removal of the 59 phosphate groups the DNA fragments enzymatically acquired a T-tail followed by T7 promoter incorporation. In vitro transcription coupled to 1st and 2nd strand synthesis was done to acquire dsDNA. ChIP-Chip in Xenopus ChIP-Chip ChIP-chip samples were prepared according to the manufac- turers protocol (Roche Nimblegen). In short, DNA yielded by 4 ChIP reactions (60 embryo equivalents) was pooled and amplified using the T7 strategy. DNA purified from de-crosslinked chromatin (i.e. input, genomic control) was also amplified. Per labeling reaction 4 mg DNA was required (post-amplification). ChIP experimental samples were labeled with Cy3 and genomic control samples with Cy5. Samples were hybridized to the NimbleGen ChIP-chip microarrays by Research and Develop- ment at Roche NimbleGen (Madison, WI, USA). Promoter Microarray Design H3K4me3-positive regions to include on the promoter micro- array design were selected based on relaxed criteria, to include as many putative positive regions while still keeping to the practical size limit of one HD2 Nimblegen microarray (,2.1 million probes). A relatively simple procedure was chosen in order to retrieve an inclusive peak set. This peak selection was only used to design the promoter microarray, not for any further comparisons. Peak detection was performed using a sliding window operation, with three different thresholds for three window lengths (3, 4 or 5 consecutive probes). These different windows were used to select relatively small high peaks, as well as somewhat broader, lower peaks. As a control, all probes were randomized per microarray and sliding window detection on the randomized probes was performed. On both real and randomized data the sliding window detection was repeated with an increasing threshold. A definitive threshold was chosen such that the number of peaks in the randomized set divided by the number of peaks in the real data, the theoretical FDR, was ,10%. All overlapping positive regions were merged and each region was extended both up- and downstream by 2 kb. In total this resulted in 27,019 putative promoter regions, the same order of magnitude as the annotated genes (27,916 JGI FilteredModels genes). The design was extended by the addition of several (partially overlapping) datasets. All 59 gene ends of JGI FilteredModels genes were added, as well a set of putative transcription start sites based on EST evidence (SJvH, W.Akhtar, RCA and GJCV, manuscript in preparation), both extended by 2 kb up- and downstream. Finally, the complete tile path of scaffold_1, scaffold_73, scaffold_130, scaffold_185, scaffold_432, scaffold_654 and scaffold_2758 was included. These regions can be used to assess genomic background in peak calling applications. The complete design consists of 1,951,339 probes, which fits on a single 2.1M Nimblegen microarray, covering a total of 46,302 contiguous sequences with a median length of ,4 kb. Peak Detection To call H3K4me3 peaks based on the three biological replicates (one genome-wide and two replicates on the promoter microarray design), TileMap [11] was chosen. In contrast to the selection of positive regions for the promoter microarray design, where an inclusive set was preferred, in this case the aim was to provide a set of high-confidence H3K4me3 peaks. As Tilemap is based on a statistical model that incorporates the replicate data, it is a more solid approach than a simple sliding window procedure. Before peak detection all probes matching more than one time to the X. tropicalis genome (JGI 4.1) were removed. The following Tilemap parameters were set: method HMM, posterior probability 0.01, expected hybridization length 10. All other parameters were left to default settings. All resulting peaks of only one probe were removed, and all peaks within 1.5 kb were merged. The peaks for TBP (9,558) were called using Tilemap with default parameters. PLoS ONE | www.plosone.org 5 January 2010 | Volume 5 | Issue 1 | e8820