Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Whole transcriptome target sequencing: profiling gene expression and alternative polyadenylation with one pipeline.

ABSTRACT: We have developed a deep sequencing method called Whole Transcriptome Target Sequencing (WTTS), which sequences the 3â ends of polyA+ RNA. This method can simultaneously profile gene expression and alternative polyadenylation. A pooled total RNA sample derived from three male and three female adult frogs was used in seven trials to develop the WTTS assay. The same sample was sequenced using RNA-seq as control. Technical replicates of the same female adult frog were analyzed using the finalized WTTS library preparation method. Biological replicates, which included 10 embryo pools collected from two families at stages 6, 8, 11, 15 and 28 were subsequently analyzed to validate the finalized WTTS method. Six embryo samples collected at stages of 6, 8 and 11 were also sequenced using RNA-seq. Therefore, this submission involved a total of 26 libraries.

ORGANISM(S): Xenopus tropicalis

SUBMITTER: Zhihua Jiang

PROVIDER: E-GEOD-74919 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Accurate Profiling of Gene Expression and Alternative Polyadenylation with Whole Transcriptome Termini Site Sequencing (WTTS-Seq).

Zhou Xiang X Li Rui R Michal Jennifer J JJ Wu Xiao-Lin XL Liu Zhongzhen Z Zhao Hui H Xia Yin Y Du Weiwei W Wildung Mark R MR Pouchnik Derek J DJ Harland Richard M RM Jiang Zhihua Z

Genetics 20160420 2

Construction of next-generation sequencing (NGS) libraries involves RNA manipulation, which often creates noisy, biased, and artifactual data that contribute to errors in transcriptome analysis. In this study, a total of 19 whole transcriptome termini site sequencing (WTTS-seq) and seven RNA sequencing (RNA-seq) libraries were prepared from Xenopus tropicalis adult and embryo samples to determine the most effective library preparation method to maximize transcriptomics investigation. We strongly ...[more]

PMID: 27098915

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Project description:During development histone modifying enzymes are required for cell identity and lineage commitment, however little is known about the regulatory origins of the epigenome during embryonic development. Here we generate a comprehensive set of embryonic epigenome reference maps, which we use to determine the extent to which maternal factors shape chromatin state in Xenopus embryos. Using Î±-amanitin to inhibit zygotic transcription, we find that the majority of H3K4me3 and H3K27me3-enriched regions form a maternally defined epigenetic regulatory space with an underlying logic of hypomethylated islands. This maternal regulatory space extends to a substantial proportion of neurula stage-activated promoters. In contrast, p300-recruitment to distal regulatory regions requires embryonic transcription at most loci. The results show that H3K4me3 and H3K27me3 are part of a regulatory space that exerts an extended maternal control well into post-gastrulation development, and highlight the combinatorial action of maternal and zygotic factors through proximal and distal regulatory sequences. We have performed ChIP-sequencing of eight histone modifications, RNA polymerase II (RNAPII) and the enhancer protein p300 at five stages of development: blastula (st. 9), gastrula (st. 10.5, 12.5), neurula (st. 16) and tailbud (st. 30). These experiments allow identification of enhancers (H3K4me1, p300), promoters (H3K4me3, H3K9ac), transcribed regions (H3K36me3, RNAPII) and repressed and heterochromatic domains (H3K27me3, H3K9me2, H3K9me3, H4K20me3). In addition we generated pre-MBT (st. 8) maps for three histone modifications (H3K4me3, H3K9ac, H3K27me3) and single-base resolution DNA methylome maps using whole genome bisulfite sequencing of blastula and gastrula (st. 9 and 10.5) embryos. To determine the maternal and zygotic contributions to chromatin state, we used alpha-amanitin to block embryonic transcription. Fertilised eggs were injected with 2.3 nl of 2.67 ng/ul alpha-amanitin and developed until the control embryos reached mid-gastrulation. Alpha-amanitin and control embryos were used for RNA-seq and ChIP-seq of RNAPII, H3K4me3, H3K27me3 and p300. For all ChIP-seq samples of the epigenome reference maps and RNAPII ChIP-seq samples of the Î±-amanitin experiments three biological replicates of different chromatin isolations of 45 embryos were pooled. Two biological replicates for H3K4me3 (Î±-amanitin injected: resp. 90 and 56 embryo equivalents (eeq); control: resp. 45 and 67 eeq), H3K27me3 (Î±-amanitin injected: resp. 90 and 180 eeq; control: resp. 45 and 202 eeq) and p300 (Î±-amanitin injected: resp. 112 and 56 eeq; control: resp. 112 and 67 eeq) ChIP-seq samples of the Î±-amanitin experiments were generated. For RNA-seq samples of the Î±-amanitin experiments RNA from 5 embryos from one biological replicate was isolated and depleted of ribosomal RNA

Dataset Information

Whole transcriptome target sequencing: profiling gene expression and alternative polyadenylation with one pipeline.

Publications

Accurate Profiling of Gene Expression and Alternative Polyadenylation with Whole Transcriptome Termini Site Sequencing (WTTS-Seq).

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