## Description

This track shows 5' cap analysis gene expression (CAGE) tags and clusters in RNA extracts from different sub-cellular localizations in multiple cell lines. A CAGE cluster is a region of overlapping tags with an assigned value that represents the expression level. The data in this track were produced as part of the ENCODE Transcriptome Project.

## 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.

To show only selected subtracks, uncheck the boxes next to the tracks that you wish to hide.

This track contains the following views:

Clusters
  Pooled clusters generated by merging clusters from individual bioreplicates using mergedBed (where two bioreplicates were available). Expression was scored individually for each replicate. The score represents the average expression score of the pooled replicates. Non-parametric Irreproducible Discovery Rate (np-IDR) was run on the normalized expression scores. The expression score is number of reads mapping to a merged cluster divided by the number of total mapped reads. A complete description of the Cluster files is located in the supplemental materials directory of our downloads page. These are currently available for downloads only in a generic bed format.
Plus and Minus Signals
  These views show the signal of the amount of overlapping CAGE reads (clusters) mapped on the forward and reverse genomic strands, respectively.
Alignments
  The Alignments view shows reads mapped to the genome and indicates where bases may mismatch. Every mapped read is displayed, i.e. uncollapsed. The alignment file follows the standard SAM format of Bowtie output. The custom tag XP can be ignored. See the Bowtie Manual for more information about the SAM Bowtie output (including other tags) and the SAM Format Specification for more information on the SAM/BAM file format. Where mapping quality is not available for this track a score of 255 is used in accordance with the SAM Format Specification. Also, where the sequence quality scores are not available, all scores are displayed as 40.
TSS and TSS HMM
  There are two different files for Transcriptional Start Sites one based on GENCODE V7 and one based on Hidden Markov Modeling. A complete description of the TSS files is located in the supplemental materials directory. This view is currently available for download only in a modified gtf format.

Metadata for a particular subtrack can be found by clicking the down arrow in the list of subtracks.

Color differences in subtracks may be set as a visual cue to distinguish between the different cell types or between annotations on the plus and minus strand.

## Methods

Cells were grown according to the approved ENCODE cell culture protocols. RNA molecules longer than 200 nt were isolated from each subcellular compartment and then were fractionated into polyA+ and polyA- fractions as described in these protocols. The CAGE tags were sequenced from the 5' ends of cap-trapped cDNAs produced using RIKEN CAGE technology (Kodzius et al. 2006; Valen et al. 2009). To create the tag, a linker was attached to the 5' end of polyA+ or polyA- reverse-transcribed cDNAs which were selected by cap trapping (Carninci et al. 1996). The first 27 bp of the cDNA were cleaved using class II restriction enzymes. A linker was then attached to the 3' end of the cDNA.

After PCR amplification, the tags were sequenced (36 bp single reads) using Illumina's Genome analyzer. Tags were mapped to the human genome (hg19) using the program delve (T. Lassmann manuscript in preparation). Delve is a new probabilistic aligner focused on giving the best possible alignment of reads to a genome rather than focusing on speed. It calculates the mapping accuracy (probability of each alignment being true or not) for each alignment. There is no set limit on the number of errors allowed and therefore the mapping rate is commonly 100%. However, for analysis it is recommended to discard alignments with low mapping qualities.

Exceptions to the above protocol are the polyA- RNA samples from K562 cytosol, K562 nucleus, and prostate whole cell which were sequenced using ABI SOLiD technology. These reads were mapped using Bowtie using default parameters. Clusters were defined as regions of overlapping CAGE reads. The expression level was computed as the number of reads making up the cluster, divided by the total number of reads sequenced, times 1 million.

## Release Notes

Release 2 has three new downloads only files per experiment (Clusters, TSS Gencode 7 and TSS HMM) and four new cell lines (A459, AG04450, BJ and SK-N-SH_RA).

Release 1 on hg19 contained the original data on hg18 that was remapped and indicated in this release as Generation 0 since that data had no replicates. If there is both old and new generation data available for a particular experiment, only the new generation data is displayed and the older data is available for download. The new data for this track was done with a different process and has standard replicate numbers. The replicate labeling in the genome browser view is a counter indicating the total number of replicates submitted. The producing lab has replicate numbers that correspond to their internal bio-replicate numbering. Where these two numbering systems conflict, both are listed in the long label of the specific track.

## Credits

These data were generated and analyzed by Timo Lassmann, Phil Kapranov, Hazuki Takahashi, Yoshihide Hayashizaki, Carrie Davis, Tom Gingeras, and Piero Carninci.

Contact: Piero Carninci at RIKEN Omics Science Center

## References

Carninci P, Kvam C, Kitamura A, Ohsumi T, Okazaki Y, Itoh M, Kamiya M, Shibata K, Sasaki N, Izawa M, et al. High-efficiency full-length cDNA cloning by biotinylated CAP trapper. Genomics. 1996 November 1; 37(3):327-336.

Kodzius R, Kojima M, Nishiyori H, Nakamura M, Fukuda S, Tagami M, Sasaki D, Imamura K, Kai C, Harbers M, et al. CAGE: cap analysis of gene expression. Nat Methods. 2006 March 1; 3(3):211-222.

Valen E, Pascarella G, Chalk A, Maeda N, Kojima M, Kawazu C, Murata M, Nishiyori H, Lazarevic D, Motti D, et al. Genome-wide detection and analysis of hippocampus core promoters using DeepCAGE. Genome Res. 2009 February; 19(2):255-265.

## Data Release Policy

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.