The ENCODE project aims to discover all the DNA sequences associated with various epigenetic features, with the reasonable expectation that these will also be functional (best tested by genetic methods). However, it is not clear how to relate these results with those from evolutionary analyses. The mouse ENCODE project aims to make this connection explicitly and with a moderate breadth. Assays identical to those being used in the ENCODE project are performed in cell types in mouse that are similar or homologous to those studied in the human project. Thus we will be able to discover which epigenetic features are conserved between mouse and human, and we can examine the extent to which these overlap with the DNA sequences under negative selection. The contribution of DNA with a function preserved in mammals versus that with a function in only one species will be discovered. The results will have a significant impact on our understanding of the evolution of gene regulation.
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Cells were grown according to the approved ENCODE cell culture protocols. Cells were lysed in RLT buffer (Qiagen RNEasy kit), and processed on RNEasy midi columns according to the manufacturer's protocol, with the inclusion of the "on-column" DNAse digestion step to remove residual genomic DNA. A quantity of 75 µgs of total RNA was selected twice with oligo-dT beads (Dynal) according to the manufacturer's protocol to isolate mRNA from each of the preparations. A quantity of 100 ngs of mRNA was then processed according to the protocol in Mortazavi et al. (2008), and prepared for sequencing on the Illumina GAIIx or HiSeq platforms according to the protocol for the ChIP-Seq DNA genomic DNA kit (Illumina). Paired-end libraries were size-selected around 200 bp (fragment length). Libraries were sequenced with the Illumina HiSeq according to the manufacturer's recommendations. Paired-end reads of 100 bp length were obtained
Reads were mapped to the reference mouse genome (version mm9 with or without the Y chromosome, depending on the sex of the cell line, and without the random chromosomes in all cases) using TopHat (version 1.3.1). TopHat was used with default settings with the exception of specifying an empirically determined mean inner-mate distance and supplying known ENSEMBL version 63 splice junctions.
Wold Group: Brian Williams, Georgi Marinov, Diane Trout, Lorian Schaeffer, Gordon Kwan, Katherine Fisher, Gilberto De Salvo, Ali Mortazavi, Henry Amrhein, Brandon King
Contacts: Georgi Marinov (data coordination/informatics/experimental), Diane Trout (informatics) and Brian Williams (experimental)
Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009;10(3):R25.
Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008 Jul;5(7):621-8.
Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics. 2009 May 1;25(9):1105-11.
Data users may freely use ENCODE data, but may not, without prior consent, submit publications that use an unpublished ENCODE dataset until nine months following the release of the dataset. This date is listed in the Restricted Until column, above. The full data release policy for ENCODE is available here.