GSE27221
GSE27221: ENCODE Genome Institute of Singapore RNA-Seq
Summary: This track is produced as part of the ENCODE Project. It shows high throughput sequencing of RNA samples from tissues or sub cellular compartments from cell lines included in the ENCODE Transcriptome subproject.  For data usage terms and conditions, please refer to http://www.genome.gov/27528022  and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf  Overall Design: 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 adapters 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 (GRCh37/hg19) excluding mitochondrion, haplotypes, randoms and chromosome Y. Mapping parameters: Strand specific mapping was done using Applied Biosystems' SOLiD alignment where all the reads were mapped to the genome, and to exon-exon junction database. Seed and extend strategy is adopted where initial seed length of 25 is mapped with maximum of 2 mismatches and then extended to read length, each color space match is awarded a score of +1 and each mismatch is awarded a penalty of -2. After extension each read is trimmed to its maximum score, shortest length. The color space sequences are then converted into base space and checked to ensure that each sequence has a maximum of 2 base pair mismatches. If any sequence has more than 2 mismatches, then that sequence is discarded.
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