Unknown,Transcriptomics,Genomics,Proteomics

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Whole transcriptome profiling of beta cell development in the mouse

ABSTRACT: Knowledge of the genetic signatures of specific pancreatic cellular populations, both during development and in the adult animal, provides an objective basis for evaluating genetic signatures of cultured human embryonic stem cells (hESCs) and reprogrammed pancreatic mouse cells. Previous transcriptome profiling studies have utilized DNA microarrays, which have well-defined limitations, and few have utilized FACS-purified cell populations. To overcome both of these limitations we utilized a series of mice with fluorescent reporter alleles (Sox17, Pdx1, Ptf1a, Ngn3, Insm1) or transgene (Ins1) to isolate three biological replicates of 12 different pancreatic progenitor or adult cell populations between embryonic day 8.0 and post-natal day 60. These RNAs were then used to make 36 libraries which were sequenced to an average depth of 50 million reads each. After mapping these reads to mouse genome using a standardized analysis pipeline the resulting datasets were analyzed in several ways.

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Mus musculus

SUBMITTER: Mark Magnuson

PROVIDER: E-MTAB-2816 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Aim: We aim to compare current (MeDIP-seq), new (Illumina Infinium 450K BeadChip) and future (PacBio) methods for whole genome DNA methylation analysis. As the interest in determination of disease methylation profiles increases, the scope, advantages and limitations of these methods requires assessment. There are key questions to answer and specific challenges to overcome. For example, how much detail/resolution is sufficient to identify regions of differential methylation and regions of biological/medical significance within a sample? How much coverage of the genome is required for accurate methylation analysis? Is it important to confirm which regions of the genome are unmethylated in addition to focusing on those that are methylated? Loss of methylation may be of equal importance within the cell since this may also contribute to disease pathogenesis. A multi-method (affinity enrichment/bisulphite-conversion based/direct sequencing of methyl-cytosine) and technology platform (Illumina HiSeq/PacBio/Illumina Infinium BeadChip) comparison will enable us to determine the strengths and weakness of each method. We propose to compare four methods using two DNA samples from the Coriell Institute for Cell Repository to assess both current and future capabilities for whole genome methylation analysis in parallel: A) MeDIP-seq using Illumina HiSeq B) Illumina Infinium HumanMethylation 450K BeadChip and C) whole genome methylation sequencing using PacBio. Existing single molecule deep bisulphite sequencing data generated previously from these same samples at the WTSI for targeted regions (30-40 genes) on the human X chromosome will be used to assess performance of each method. The methods selected for this study will generate data covering a range of resolutions from a whole genome scan to array (target defined) resolution and up to single base pair, single molecule resolution; the highest level of detail possible with methods currently available.Samples: DNA from sibling pair GM01240 (female) and GM01240 (male).Requirements: Both samples will be analysed using;A.MeDIP-seq using Illumina HiSeq (one HiSeq lane, 75bp paired end, per sample) B.Illumina Infinium HumanMethylation 450K BeadChipWe are expecting a potentially unnecessary high coverage using one HiSeq lane per sample. However, for the MeDIP procedure we do not have a multiplexing procedure in place. Our requirements for PacBio sequencing have been discussed with and will be supported by the Sequencing Technology Development group.

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Whole transcriptome profiling of beta cell development in the mouse

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