Unknown,Transcriptomics,Genomics,Proteomics

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IRNA-seq: Computational method for genome wide assessment of acute transcriptional regulation from total RNA-seq data

ABSTRACT: RNA-seq is a sensitive and accurate technique to compare steady state levels of RNA between different cellular states. However, as it does not provide an account of transcriptional activity per se, other technologies are needed to more precisely determine acute transcriptional responses. Here, we have developed an easy, sensitive and accurate novel method, iRNA-seq, for genome-wide assessment of transcriptional activity based on analysis of intron coverage from total RNA-seq data. To test our method, we have performed total RNA-seq and RNA polymerase II (RNAPII) ChIP-seq profiling of the acute transcriptional response of human adipocytes to TNFM-NM-1 treatment and analyzed these using iRNA-seq in addition to different publically availbale dataset. Comparison of the results derived from iRNA-seq analyses with results derived using current methods for genome-wide determination of transcriptional activity, i.e. Global Run-On (GRO)-seq and RNA polymerase II (RNAPII) ChIP-seq, demonstrate that iRNA-seq provides very similar results in terms of number of regulated genes and their fold change. However, unlike the current methods that are all very labor-intensive and demanding in terms of sample material and technologies, iRNA-seq is cheap and easy and requires very little sample material. In conclusion, iRNA-seq offers an attractive novel alternative to current methods for determination of changes in transcriptional activity at a genome-wide level. Genome-wide assesment of the acute transcriptional response to TNFa in human SGBS adiposytes using total RNA-seq data end RNAPII ChIP-seq

ORGANISM(S): Homo sapiens

SUBMITTER: Susanne Mandrup

PROVIDER: E-GEOD-60462 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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iRNA-seq: computational method for genome-wide assessment of acute transcriptional regulation from total RNA-seq data.

Madsen Jesper Grud Skat JG Schmidt Søren Fisker SF Larsen Bjørk Ditlev BD Loft Anne A Nielsen Ronni R Mandrup Susanne S

Nucleic acids research 20150106 6

RNA-seq is a sensitive and accurate technique to compare steady-state levels of RNA between different cellular states. However, as it does not provide an account of transcriptional activity per se, other technologies are needed to more precisely determine acute transcriptional responses. Here, we have developed an easy, sensitive and accurate novel computational method, IRNA-SEQ: , for genome-wide assessment of transcriptional activity based on analysis of intron coverage from total RNA-seq data ...[more]

PMID: 25564527

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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

IRNA-seq: Computational method for genome wide assessment of acute transcriptional regulation from total RNA-seq data

Publications

iRNA-seq: computational method for genome-wide assessment of acute transcriptional regulation from total RNA-seq data.

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