Embryonic forebrain, midbrain and limb tissue was isolated from mouse embryos at E11.5. Cross-linking, chromatin isolation, sonication and immuno-precipitation using an anti-p300 antibody were performed as previously described40 (link),46 (link). ChIP DNA was further sheared by sonication, end-repaired, ligated to sequencing adapters and amplified by emulsion PCR as previously described47 (link). Gel-purified amplified ChIP DNA between 300 and 500 bp was sequenced on the Illumina Genome Analyzer II platform to generate 36-bp reads.
Sequence reads were aligned to the mouse reference genome (mm9) using BLAT48 (link). Uniquely aligned reads were extended to 300 bp in the 3′ direction and used to determine the read coverage at individual nucleotides at 25-bp intervals throughout the mouse genome. p300-enriched regions (peaks) with an estimated FDR of ≤ 0.01 were identified by comparison with a random distribution of the same number of reads. Candidate peaks mapping to repetitive regions were removed as probable artefacts.
Candidate regions for transgenic testing were selected based on ChIP-seq results and cover a wide spectrum of conservation. Enhancer candidate regions were amplified by PCR from human genomic DNA and cloned into an Hsp68-promoter-LacZ reporter vector as previously described6 (link),31 (link). Transgenic mouse embryos were generated and evaluated for reproducible LacZ activity at E11.5 as previously described6 (link).
Total RNA from E11.5 whole embryos and forebrain tissue was hybridized to GeneChip Mouse Genome 430 2.0 arrays (Affymetrix) and analysed according to the manufacturer's recommendations. Forebrain- and whole-embryo-enriched genes were identified as having at least 2.5-fold greater expression in one data set compared with the other, and a minimum signal intensity of 100. Limb-enriched genes were identified by comparison with publicly available wild-type E11.5 proximal hindlimb gene expression data (Gene Expression Omnibus (GEO) series GSE10516, samples GSM264689, GSM264690 and GSM264691)49 (link).
Sequence reads were aligned to the mouse reference genome (mm9) using BLAT48 (link). Uniquely aligned reads were extended to 300 bp in the 3′ direction and used to determine the read coverage at individual nucleotides at 25-bp intervals throughout the mouse genome. p300-enriched regions (peaks) with an estimated FDR of ≤ 0.01 were identified by comparison with a random distribution of the same number of reads. Candidate peaks mapping to repetitive regions were removed as probable artefacts.
Candidate regions for transgenic testing were selected based on ChIP-seq results and cover a wide spectrum of conservation. Enhancer candidate regions were amplified by PCR from human genomic DNA and cloned into an Hsp68-promoter-LacZ reporter vector as previously described6 (link),31 (link). Transgenic mouse embryos were generated and evaluated for reproducible LacZ activity at E11.5 as previously described6 (link).
Total RNA from E11.5 whole embryos and forebrain tissue was hybridized to GeneChip Mouse Genome 430 2.0 arrays (Affymetrix) and analysed according to the manufacturer's recommendations. Forebrain- and whole-embryo-enriched genes were identified as having at least 2.5-fold greater expression in one data set compared with the other, and a minimum signal intensity of 100. Limb-enriched genes were identified by comparison with publicly available wild-type E11.5 proximal hindlimb gene expression data (Gene Expression Omnibus (GEO) series GSE10516, samples GSM264689, GSM264690 and GSM264691)49 (link).
