Description
This track is produced as part of the ENCODE Project. This track shows short tag
sequencing of cDNA obtained from biological replicate samples (different culture
plates) of the ENCODE cell lines. The sequences were aligned to the human genome
(hg18) and UCSC known-gene splice junctions using different sequence alignment
programs such ELAND (Illumina) or Bowtie (Langmead et al., 2009). RNA-seq is a
method for mapping and quantifying the transcriptome of any organism that has a
genomic DNA sequence assembly. RNA-seq is performed by reverse-transcribing an
RNA sample into cDNA, followed by high throughput DNA sequencing, which was done
here on an Illumina Genome Analyzer (GA2) (Mortazavi et al., 2008). The
transcriptome measurements shown on these tracks were performed on polyA selected
RNA from total cellular RNA. Data have been produced in two formats: single reads,
each of which comes from one end of a randomly primed cDNA molecule; and
paired-end reads, which are obtained as pairs from both ends cDNAs resulting from
random priming. The resulting sequence reads are then informatically mapped onto
the genome sequence (Alignments). Those that don't map to the genome are mapped
to known RNA splice junctions (Splice Sites). These mapped reads are then counted
to determine their frequency of occurrence at known gene models. Sequence reads
that cluster at genome locations that lack an existing transcript model are also
identified informatically and they are quantified. RNA-seq is especially suited
for giving information about RNA splicing patterns and for determining
unequivocally the presence or absence of lower abundance class RNAs. As
performed here, internal RNA standards are used to assist in quantification and
to provide internal process controls. This RNA-seq protocol does not specify the
coding strand. As a result, there will be ambiguity at loci where both strands
are transcribed. The "randomly primed" reverse transcription is, apparently, not
fully random. This is inferred from a sequence bias in the first residues of the
read population, and this likely contributes to observed unevenness in sequence
coverage across transcripts.
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:
- RPKM
- RefSeq gene models are displayed shaded by their RPKM (Reads Per Kilobase of
exon per Million reads) value. RPKM is reported in the score of each element, and
each element
is shaded using a gray scale that becomes darker as RPKM increases. The RPKM
measure assists in visualizing the relative amount of a given transcript
across multiple samples.
- Alignments
- The Alignments view shows reads mapped to the genome.
Alignments are colored by cell type.
Methods
Gene expression is measured in Reads Per Kilobase exon per Million reads
(RPKM; Mortazavi et al., 2008). RNA-seq reads are aligned to RefSeq gene models.
RPKM is then calculated by dividing the total number of reads that align to the
gene model (RefSeq) by the size of the spliced transcript in kilobases. This
number is then divided by the total number of reads in millions for the
experiment. For example, if x reads align to a RefSeq gene whose spliced
transcript is y kb in size and there are z million reads in the experiment,
then RPKM = x/(y*z).
Cells were grown according to the approved ENCODE cell culture protocols. A total of 2 X 107 cells were
lysed in either 4mls of RLT buffer (Qiagen RNEasy kit), and processed on 2
RNEasy midi columns according to the manufacturer's protocol, with the inclusion
of the "on-column" DNAse digestion step to remove residual genomic DNA. 75 �g
of total RNA was selected twice with oligodT beads (Dynal) according to the
manufacturer's protocol to isolate mRNA from each of the preparations. 100 ng of
mRNA was then processed according to the protocol in Mortazavi et al. (2008),
and prepared for sequencing on the Genome Analyzer flow cell according to the
protocol for the ChIPSeq DNA genomic DNA kit (Illumina).
Following alignment of the sequence reads to the genome assembly as described
above, the sequence reads were further analyzed using the ERANGE 3.0 software
package, which quantifies the number of reads falling within the mapped boundaries
of known transcripts from the Gencode annotations. ERANGE assigns both genomically
unique reads and reads that occur in 2-10 genomic locations for quantification.
Verification
- Known exon maps as displayed on the genome browser are confirmed by the
alignment of sequence reads.
- Known spliced exons are detected at the expected frequency for transcripts
of given abundance.
- RT-QCPR confirms expression measurements with r > 0.8
Credits
Myers Group: Florencia Pauli, Tim Reddy
Wold Group: Ali Mortazavi, Brian Williams, Diane Trout, Brandon King, Ken McCue,
Lorian Schaeffer.
Illumina gene expression group: Gary Schroth, Shujun Luo, Eric Vermaas.
Contacts:
Tim Reddy
and
Flo Pauli
(experimental).
References
Mortazavi A, Williams BA, McCue K, Schaeffer L, and Wold BJ.
Mapping and
quantifying mammalian transcriptomes by RNA-Seq
Nature Methods. 2008 Jul; 5(7):621-628.
Langmead B, Trapnell C, Pop M, Salzberg SL.
Ultrafast and
memory-efficient alignment of short DNA sequences to the human genome
Genome Biology. 2009 Mar; 10:R25.
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.
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