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RNA-seq from ENCODE/Stanford/Yale


ABSTRACT: The tracks show enrichment of RNA sequence tags mapped to the mouse genome generated by high throughput sequencing (RNA-Seq). Double stranded cDNA was synthesized from enriched RNA that was obtained after depletion of ribosomal RNA. Pieces of cDNA, 300-350 nucleotides in length, were PCR amplified, adapter ligated, and sequenced on an Illumina HiSeq sequencer. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Cells were grown according to the approved ENCODE cell culture protocols (http://hgwdev.cse.ucsc.edu/ENCODE/protocols/cell). Total RNA was extracted using RNeasy Mini Kit (74104, Aiagen), following the manufacturer's protocol. Ribosomal RNA was removed from total RNA using the Ribo-ZeroTM Gold Kits (MRZG126, Epicentre). Double-stranded cDNA synthesis was performed on the rRNA depleted RNA using random primers and the SuperScript double-stranded cDNA synthesis kit (11917-010, Life Tech). After first strand cDNA synthesis, NucAway Spin Column (Ambion cat. 100070-30) was used to remove dNTPs. In the second strand cDNA synthesis reaction, dTTP in the dNTP mix was substituted with dUTP. After end repair and addition of 'A' base to 3' end, illumina paired-end adapter was ligated to Double-stranded cDNA library. After gel size selection of adapter ligated cDNA (300-350), Uracil-N-Glycosylase (UNG: Applied Biosystems) was used to digest the second strand cDNA (Parkhomchuk et al. , 2009). PCR amplified adapter ligated cDNA was sequenced using Illumina HiSeq. Sequence reads of 2x101 nt long with 0-2 mismatches were mapped to the mouse genome (version mm9) using the BWA aligner, version 0.5.7. The signal height corresponds to the number of overlapping fragments at each nucleotide position in the genome.

ORGANISM(S): Mus musculus

PROVIDER: E-GEOD-39619 | BioStudies |

REPOSITORIES: biostudies

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