## Description

Genome wide methylation (MeDIP-seq and MRE-seq), histone H3 lysine 4 trimethylation (H3K4me3) and gene expression (RNA-seq and RNA-seq (SMART)) data were generated from postmortem human frontal cortex gray matter of a 57 year-old male. This was done to investigate the role that intragenic, tissue-specific CpG island methylation plays in controlling gene expression (Maunakea, et al. 2010).

## Display Conventions and Configuration

This track is a multi-view composite track that contains multiple data types (views). For each view, there are multiple subtracks that display individually on the browser. Instructions for configuring multi-view tracks are here. The following views are in this track:

Raw Singal
  Density graph (wiggle) of signal enrichment.
CpG score
  DNA methylation score on CpG sites.

## Methods

DNA, RNA and native chromatin were extracted using standard methods; assay specific methods are described below. MRE-seq, MeDIP-seq, H3K4me3 ChIP-seq, RNA-seq and RNA-seq (SMART) libraries were sequenced using an Illumina Genome Analyzer II. Sequencing reads are available through the NCBI SRA (study accession number SRP002318).

### MeDIP-seq (Methylated DNA immunoprecipitation and sequencing)

MeDIP-seq uses immunoprecipitation to extract the methylated fraction of the genome. Purified DNA was first sheared and processed following the Illumina Genomic DNA Library Kit protocol. These DNA fragments were then immunoprecipitated using an antibody raised against 5-methylcytosine, the methylated form of cytosine, before constructing a library, which was sequenced and mapped to the genome.

### MRE-seq (Methyl-sensitive restriction enzyme digest and sequencing)

MRE-seq identifies unmethylated CpG sites by sequencing size-selected fragments from parallel DNA digestions with the MREs HpaII, Hin6I, and AciI. Since these enzymes require unmethylated CpG sites within their recognition sequences to cut DNA, identifying the end of each fragment generated allows inference of a single unmethylated cytosine. The 3 digests were combined and size-selected by gel electrophoresis to enrich for unmethylated CpG sites in close proximity. A library was constructed and sequenced; the sequence reads were then mapped to the genome with the additional requirement that they map to a known MRE recognition site.

### H3K4me3 ChIP-seq (Histone H3 lysine 4 trimethylation chromatin immunoprecipitation and sequencing)

Chromatin immunoprecipitation was performed to enrich for histone H3 modified at lysine position 4 with trimethylation (H3K4me3), as this histone modification is associated with promoters. A ChIP-seq library was constructed as described in Robertson, et al. 2007, sequenced and mapped to the genome.

### RNA-seq and RNA-seq SMART (RNA sequencing and SMART-tagged RNA sequencing)

Sheared RNA was used to synthesize full-length single-stranded cDNAs as described by Morin, et al. 2008. A library was constructed and sequenced, and sequence reads are mapped to the genome. The 5' end of transcripts were tagged with a sequence tag, called a "SMART tag", while making cDNA library for sequencing. SMART tagged reads were used to infer transcription initiation, while all reads together are used to infer gene expression level.

## Verification

### MeDIP-seq

Each post-amplification library was QC'd for quantity, quality and size distribution by spectrophotometry and Agilent DNA Bioanalyzer analysis. Four independent PCR reactions were performed to confirm enrichment for methylated and de-enrichment for unmethylated sequences, compared to input sonicated DNA. Visual inspection of extended coverage browser tracks confirmed expectations: lack of MeDIP signal in most 5' CpG island promoters and in regions devoid of CpG sites, as well as high MeDIP signal at known methylated sites (i.e. some imprinted regions).

### MRE-seq

Each post-amplification library was QC'd for quantity, quality and size distribution by Nanodrop spectrophotometry and Agilent DNA Bioanalyzer analysis. Prior to high-throughput sequencing, a portion of each library was cloned into a sequencing vector and ~24 individual clones were Sanger sequenced to confirm the presence of MRE sites at the ends of each insert. Illumina sequencing reads were filtered to only include those that map to MRE sites in the reference. MRE reads occured frequently in 5' CpG islands, which are often unmethylated and are enriched for the MRE recognition sequences relative to rest of the genome.

### H3K4me3 ChIP-seq

Each post-amplification library was examined for quantity, quality and size distribution by Nanodrop spectrophotometry, Qubit fluoremetry and Agilent DNA Bioanalyzer. Fold H3K4me3 enrichment was confirmed by comparison to non-specific rabbit IgG enrichment. Visual inspection of the browser track of called peaks confirmed enrichment at a subset of annotated promoters.

### RNA-seq and RNA-seq (SMART)

Each post-amplification library was examined for quantity, quality and size distribution by Nanodrop spectrophotometry, Qubit fluoremetry and Agilent DNA Bioanalyzer. Visual inspection of the browser track of extended reads confirmed enrichment at annotated exons and UTRs. SMART-tagged reads were enriched at known promoters, as expected.

## Credits

UCSF: Joseph Costello, Raman Nagarajan, Shaun Fouse, Brett Johnson, Chibo Hong, Ksenya Shchors, Vivi M. Heine, David H. Rowitch

Genome Sciences Centre, BC Cancer Agency: Mikhail Bilenky, Cletus D'Souza, Cydney Nielsen, Yongjun Zhao, Allen Delaney, Richard Varhol, Nina Thiessen, Steven S.J. Jones, Marco A. Marra, Martin Hirst

Washington University, St. Louis, MO: Ting Wang, Xiaoyun Xing, Chris Fiore, Maximiliaan Schillebeeckx

UCSC: Tracy J. Ballinger, David Haussler

McGill: Gustavo Turecki

## References

Maunakea AK, Nagarajan RP, Bilenky M, Ballinger TJ, D'Souza C, Fouse SD, Johnson BE, Hong C, Nielsen C, Zhao Y, et al. Conserved Role of Intragenic DNA Methylation in Regulating Alternative Promoters. Nature. 2010 Jul 8;466:253-7.

Robertson G, Hirst M, Bainbridge M, Bilenky M, Zhao Y, Zeng T, Euskirchen G, Bernier B, Varhol R, Delaney A, et al. Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods. 2007;4(8):651-57.

Morin RD, Bainbridge M, Fejes A, Hirst M, Krzywinski M, Pugh TJ, McDonald H, Varhol R, Jones SJM, Marra MA. Profiling the HeLa S3 transcriptome using randomly primed cDNA and massively parallel short-read sequencing. Biotechniques. July 2008;45(1):81-94.