Transcriptome profiling of zebrafish neural crest mutants
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2000
ORGANISM(S): Danio rerio
SUBMITTER:
PROVIDER: E-ERAD-203 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Total RNA was extracted by Dr Joan Heath's team from morpholigcally abnormal and wildtype sibling zebrafish larvae of intestinal mutants initially identified in the Liverplus-Screen (Ober EA et al Nature 2006 PMID:16799568). The mutants have ZFIN Identifiers tti s450, set s453 and puck s456 (s: San Francisco; Dr Didier Stainier Lab). The 3' end of fragmented RNA was pulled down using polyToligos attached to magnetic beads, reverse transcribed, made into Illumina libraries and sequenced using IlluminaHiSeq paired-end sequencing. Protocol: Total RNA obtained was DNase treated. Chemically fragmented RNA was enriched for the 3' ends by pull down using an anchored polyToligo attached to magnetic beads. An RNA oligo comprising part of the Illumina adapter 2 was ligated to the 5' end of the captured RNA and the RNA was eluted from the beads. Reverse transcription was primed with an anchored polyToligo with part of Illumina adapter 1 at the 5' end followed by 4 random bases, then an A, C or G base, the one of twelve 5 base indexing tags and 14T bases. An Illumina library with full adapter sequence was produced by 15 cycles of PCR. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
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Project description:Total RNA was extracted from morpholically abnormal and sibling wild type embryos identified by the Zebrafish Mutation Project (http://www.sanger.ac.uk/Projects/D_rerio/zmp/). The 3 end of fragmented RNA was pulled down using polyToligos attached to magnetic beads, reverse transcribed, made into Illumina libraries and sequenced using IlluminaHiSeq paired-end sequencing. Protocol: Total RNA was extracted from mouse embryos using Trizol and DNase treated. Chemically fragmented RNA was enriched for the 3 ends by pulled down using an anchored polyToligo attached to magnetic beads. An RNA oligo comprising part of the Illumina adapter 2 was ligated to the 5 end of the captured RNA and the RNA was eluted from the beads. Reverse transcription was primed with an anchored polyToligo with part of Illumina adapter 1 at the 5 end followed by 4 random bases, then an A, C or G base, then one of twelve5 base indexing tags and 14 T bases. An Illumina library with full adapter sequence was produced by 15 cycles of PCR. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
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