This track is produced as part of the ENCODE Transcriptome Project. It shows high throughput sequencing of RNA samples from tissues or sub cellular compartments from cell lines included in the ENCODE Transcriptome subproject. The overall goal of the ENCODE project is to identify and characterize all functional elements in the sequence of the human genome.
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The RNA-Seq data were generated from high quality polyA RNA, and the RNA-Seq libraries were constructed using SOLiD Whole Transcriptome (WT) protocol and reagent kit. Total RNA in good quality was used as starting materials and purified twice through MACs polyT column aimed to enrich polyA and remove any contaminants (e.g., rRNA, tRNA, DNA, protein etc.). A one microgram enriched polyA RNA sample was then fragmented to small pieces, and a gel-based selection method was performed to collect fragmented random polyA at a size-range of 50-150 nt in length. The collected fragmental RNA was then hybridized and ligated to a mix of adaptors provided from ABI, followed by reverse transcription to generate corresponding cDNAs. The resulting cDNA library was further amplified by PCR and sequenced by SOLiD platform for single reads at 35 bp length (new version in 50 bp length). Cells were grown according to the approved ENCODE cell culture protocols.
Data: The SOLiD-generated RNA-Seq reads were 35 bp in length. An initial filtering process was performed to remove any non-desirable contamination sequences, such as rRNA, tRNA, and repeats etc. A read-split mapping approach was developed to map the 35 bp reads onto the reference genome (NCBI Build 36/hg18). Specifically, the 35 bp reads were divided into two parts (1st-25 bp and 2nd-25 bp with 10 bp overlapping) and mapped separately. An extension mapping analysis was further performed to generate score counts from each read and use the score numbers as a gauge of filtering reference (e.g., scoring >26 was used). The reads with mapping locations N≤1 or N>10 were excluded from further analysis. As a unique strand-specific feature from the SOLiD RNA-Seq, the data sets generated by SOLiD RNA-Seq were strand-specific and mapped on exons with strand-specificity.
Mapping parameters: Mapping was done using Applied Biosystems' SOLiD alignment for whole transcriptome analysis pipeline. Two mismatches were allowed in the 25 bp color space seed sequence with progressive alignment performed to find the full mapping location. A score is computed for each mapping location and any location that scored ≤26 was filtered.
The GIS RNA-seq libraries and sequence data for transcriptome analysis were produced at the Genome Institute of Singapore. The data were mapped and analyzed by scientists from the Genome Institute of Singapore.
Contact: RUAN Xiaoan
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