GSE35584
GSE35584: RNA-seq from ENCODE/HudsonAlpha
Summary:                 This track is produced as part of the ENCODE Project. RNA-seq is a method for mapping and quantifying the transcriptome of any organism that has a genomic DNA sequence assembly (Mortazavi et al., 2008). Biological replicates of ENCODE cell lines were grown on separate culture plates, total RNA was purified and polyA selected two times. mRNA was then fragmented by magnesium-catalyzed hydrolysis, reverse transcribed to cDNA by random priming and amplified. The cDNA was sequenced on an Illumina Genome Analyzer (GAI or GAIIx).  The DNA sequences were aligned to the NCBI Build37 (hg19) version of the human genome using the sequence alignment programs ELAND (Illumina) or Bowtie (Langmead et al., 2009). The first 10 residues of sequencing have a weak characteristic nucleotide bias of unknown origin. This RNA-seq protocol does not specify the coding strand. As a result, there will be ambiguity at loci where both strands are transcribed.  This is the first NCBI Build37 (hg19) release of this track (Jan 2012). This release includes the 3 datasets (Jurkat, A549/DEX100nm, and A549/EtOH2pct) previously released on NCBI Build36 (hg18) and adds data for several more cell types and growth conditions in replicate. Four types of download files are available for each replicate including the Raw Data (fastq), Transcripts GencodeV7 (gtf), Raw Signal (bigwig), and Alignments (bam).  Overall Design: Experimental ProceduresCells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell) except for H1-hESC for which frozen cell pellets were purchased from Cellular Dynamics. Cells were lysed in RLT buffer (Qiagen RNEasy kit) and processed on RNEasy midi columns according to the manufacturer's protocol, with the inclusion of the "on-column" DNase digestion step to remove residual genomic DNA. mRNA was isolated from at least 10 ug of total RNA with oligo(dT) two times (Dynabeads mRNA PurificationgKit, Invitrogen). Alternatively, cells were lysed and mRNA was purified directly two times with oligo(dT) (Dynabeads mRNA DIRECT Kit, Invitrogen). 100 ng of mRNA was fragmented by magnesium-catalyzed hydrolysis and reverse transcribed to cDNA by random priming according to the protocol in Mortazavi et al. (2008). cDNA was prepared for sequencing on the Genome Analyzer flowcell according to the protocol for the ChIPSeq DNA genomic DNA kit (Illumina). The sequencing libraries were size-selected around 225 bp and amplified with 15 rounds of PCR.Libraries were sequenced with an Illumina Genome Analyzer I or an Illumina Genome Analyzer IIx according to the manufacturer's recommendations. Single end reads of 36 nt in length were obtained.Data Processing and AnalysisFastq files were made from qseq files generated by the Illumina pipeline (Casava 1.7). The Raw Signal files (bigWig) were generated from bedgraph files and the score was calculated as the number of reads at that position divided by the total number of reads divided by one million.Casava export files were aligned to the NCBI Build37 (hg19) version of the human genome with ELAND (Illumina), generating SAM files. Fastq files of experiments that were previously aligned to NCBI Build36 (hg18) were aligned to NCBI Build37 (hg19) using Bowtie (Langmead et al., 2009; parameters: -S -n 2 -k 11 -m 10 --best), also generating SAM files. SAM files were converted to BAM with SAMtools (Li et al., 2009).Gene expression within Gencode.v7 (Harrow et al., 2006) gene models was estimated using Cufflinks v0.9.3 (Roberts et al., 2011). Estimates of transcript abundance were reported in Fragments Per Kilobase of exon per Million fragments mapped (FPKM). FPKM is calculated by dividing the total number of fragments that align to the gene model by the size of the spliced transcript (exons) in kilobases. This number is then divided by the total number of reads in millions for the experiment. FPKM is reported in the last column of the gtf (TranscriptGencV7) files.Raw Data (fastq), Raw Signal (bigWig), Alignments (bam) and Transcript Gencode V7 (gtf) files are available from the Downloads (http://hgwdev.cse.ucsc.edu/cgi-bin/hgFileUi?g=wgEncodeHaibRnaSeq) page.