## Description

The tracks show enrichment of RNA sequence tags generated by high throughput sequencing (RNA Seq) and mapped to the human genome. Double stranded cDNA was synthesized either from polyadenylated RNA (polyA+) or, in order to analyze transcription from RNA polymerase III, from RNA remaining after depletion of both ribosomal RNA and polyadenylated RNA (polyA-/rRNA-). PCR amplified, adapter ligated cDNA, 150-300nt long, was sequenced on an Illumina GA sequencer.

Where designated, cell lines received specific treatments prior to RNA isolation. As indicated, K562 cells were treated with either interferon-a or interferon-g for 30 minutes or 6 hours. These experiments were carried out in conjunction with ChIP-Seq experiments on the transcription factors STAT1 and STAT2 in order to examine the effects that inducers of a specific transcriptional response might have on gene expression and on transcription factor binding site discovery. K562 cells were treated with a-amanitin in order to examine the effects of RNA polymerase II inhibition on RNA polymerase III-mediated transcription.

## 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. This track contains the following views:

Signal
  Density graph (wiggle) of signal enrichment based on processed data.
Alignments
  Mappings of short reads to the genome.

## Methods

Cells were grown according to the approved ENCODE cell culture protocols. Total RNA was extracted using TRIzol reagents (15596-018, Life Tech), following the manufacturer's protocol. For polyA+ samples, polyadenylated RNA was purified using the MicroPoly(A) Purist kit (AM1919, Life Tech) and fragmented using RNA Fragmentation Reagent (AM8740, Life Tech). Illumina adapters were ligated to double stranded cDNA which was synthesized using reagents from Life Tech (11917-010). For polyA-/rRNA- samples, ribosomal RNA was removed from total RNA using the RiboMinus Eukaryote Kit (K1550-02, Life Tech) and polyadenylated RNA was purified away from the rRNA depleted fraction using the MicroPoly(A) Purist kit (AM1919, Life Tech). Double-stranded cDNA synthesis was performed on the rRNA/polyA depleted RNA using random primers and the SuperScript double-stranded cDNA synthesis kit (11917-010, Life Tech).

PCR amplified adapter ligated cDNA (150-300 bp) was sequenced using Illumina GA. Sequence reads of 27-33nt long with 0-2 mismatches were mapped to the genome. The signal height corresponds to the number of overlapping fragments at each nucleotide position in the genome. Samples originally mapped to the hg18 version of the human genome were remapped to hg19 using the BWA aligner, version 0.5.7.

## Credits

These data were generated and analyzed by the labs of Michael Snyder, and Sherman Weissman at Yale University;

Contact: Philip Cayting.

## References

Nagalakshmi U, Zhong Wang , Waern K, Shou C, Raha D, Gerstein M, Snyder M The transcriptional landscape of the yeast genome defined by RNA sequencing. Science. 2008 Jun 6;320(5881):1344-1349.

Raha D, Wang Z, Moqtaderi Z, Wu L, Zhong G, Gerstein M, Struhl K, and Snyder M Close association of RNA polymerase II and many transcription factors with Pol III genes. Proc Natl Acad Sci. 2010 Feb 23;107(8):3639-44. Epub 2010 Feb 5.

Wu JQ, Habegger L, Noisa P, Szekely A, Qiu C, Hutchison S, Raha D, Egholm M, Lin H, Weissman S, Cui W, Gerstein M, Snyder M Dynamic Transcriptomes during Neural Differentiation of Human Embryonic Stem Cells Revealed by Integrating Short, Long, and Paired-end Sequencing. Proc Natl Acad Sci. 2010 March 1 (Epub ahead of publication).

## 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 on the track configuration page and the download page. The full data release policy for ENCODE is available here.