Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

GENCODE PCR-Seq Batch XI

ABSTRACT: As part of the ENCODE consortium the GENCODE project is producing a reference gene set through manual and automated gene prediction. Selected transcript models are verified experimentally by RT-PCR amplification of at least one of their unique splice junctions followed by sequencing. The experiment targets are manually annotated transcripts with novel or putative status, non-pseudogene biotype and unique splice junctions not validated previously. For Batch XI 966 splice junctions from GENCODE13 (released July 2012) and 640 splice junctions from GENCODE14 (released October 2012) were chosen for experimental verification.

ORGANISM(S): Homo sapiens

SUBMITTER: Jose Gonzalez

PROVIDER: E-MTAB-1505 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome.

Howald Cédric C Tanzer Andrea A Chrast Jacqueline J Kokocinski Felix F Derrien Thomas T Walters Nathalie N Gonzalez Jose M JM Frankish Adam A Aken Bronwen L BL Hourlier Thibaut T Vogel Jan-Hinnerk JH White Simon S Searle Stephen S Harrow Jennifer J Hubbard Tim J TJ Guigó Roderic R Reymond Alexandre A

Genome research 20120901 9

Within the ENCODE Consortium, GENCODE aimed to accurately annotate all protein-coding genes, pseudogenes, and noncoding transcribed loci in the human genome through manual curation and computational methods. Annotated transcript structures were assessed, and less well-supported loci were systematically, experimentally validated. Predicted exon-exon junctions were evaluated by RT-PCR amplification followed by highly multiplexed sequencing readout, a method we called RT-PCR-seq. Seventy-nine perce ...[more]

PMID: 22955982

Publication: 1/2

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Project description:We used all five current human African trypanosomiasis drugs for genome-scale RNA interference (RNAi) target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate anti-trypanosomal drug action. RIT-seq profiling links more than fifty additional genes to drug action. This data has been described in the following article [doi:10.1038/nature10771] and its further analysis can be freely submitted for publication. For information on the proper use of data shared by the Wellcome Trust Sanger Institute (including information on acknowledgement), please see http://www.sanger.ac.uk/datasharing/. Protocol - Assembly of the bloodstream-form T. brucei RNAi library and the RNAi target sequencing (RIT-seq) approach were reported previously (Alsford, S. et al. High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res 21, 915-924, 264 doi:gr.115089.110 [pii] 265 10.1101/gr.115089.110 (2011). Briefly, a tetracycline-inducible RNAi plasmid library, containing randomly sheared genomic fragments (mean ~600 bp) under the control of head-to-head T7 promoters, was targeted to a single genomic locus that had been validated for robust expression. For this study, the library was grown under inducing conditions with drug selection and genomic DNA was isolated from surviving populations. For chemical RIT-seq profiling, adapter-ligated sequencing libraries were prepared from each genomic DNA sample and used to amplify DNA fragments containing RNAi cassette-insert junctions in semi-specific PCR reactions; one primer was specific for the RNAi vector and the other for the Illumina adapter. Size-selected DNA was sequenced with 76 cycle paired-end runs on an Illumina GAII. Illumina sequencing reads containing a nine-base RNAi vector junction sequence were then mapped to the T. brucei reference genome. Where loss of function increases drug tolerance, RNAi-target sequence representation is increased relative to the otherwise susceptible population, revealing hot spots.

Dataset Information

GENCODE PCR-Seq Batch XI

Publications

Combining RT-PCR-seq and RNA-seq to catalog all genic elements encoded in the human genome.

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