National Science Foundation
Division of Biological Infrastructure
Plant Genome Research Program
Exploiting Chromatin Landmarks to Characterize Complex Plant Genomes, Project DBI-0922447
Steve van Nocker, PI vannocke@msu.edu
Data Access and Annotation: M. x domestica cv. Golden Delicious flower bud and open-pollinated seedling leaf
NCBI SRA Study SRP003265
18 Aug 2010 Chromatin-tagged sequence: M. x domestica cv. Golden Delicious flower bud [NCBI SRA Report SRX025560]
Experiment design: 454 sequencing of histone-modification-specific ChIP selected DNA.
Study Summary: Optimization of ChIP-seq for the characterization of expressed genes within bulk chromatin derived from apple (Malus x domestica)
Abstract: The goal of this study was to apply histone-modification-specific ChIP-seq for the identification of weakly expressed and tissue-specific expressed genes from total genomic DNA in domestic apple (Malus x domestica). We first determined if selected genes predicted to show strong tissue-specificity of expression could be recovered through ChIP using modification-specific antibodies. We targeted known sequences representing apple genes most closely related to Arabidopsis SVP, SOC1, TFL1, AP1, and AG. Using seedling tissues, we carried out extensive optimizations of crosslinking duration, sonication times and intensities, and antibody concentrations. Four of these sequences (SVP-like, SOC1-like, TFL1-like, and AP1-like) amplified selectively from the IP. An apple ACTIN sequence, which we know from EST source analysis to be expressed strongly in numerous tissues, was amplified only after extensive cycling, and did not show enrichment in the IPs. We then scaled up the ChIP using 10 g of seedling tissues and undertook a proof-of-concept experiment with ChIP-seq runs from apple flower bud and leaf. Of ~109,000 sequences from flower bud, ~21,000 matched 7,700 known apple contig/singleton sequences (BLASTN <1E-05), and ~8,800 matched ~4,000 Arabidopsis genes (TBLASTN <1E-05). Of ~226,000 sequences from leaf, ~72,100 matched ~14,300 known apple contig/singletons, and ~31,000 matched ~7,000 Arabidopsis genes. Given the abundance of unmatched, expressed-gene-like sequences in this dataset, we concluded that these limited preliminary experiments were highly successful in identifying previously unknown apple genes.
Contact Name: Steven van Nocker
Contact E-mail: vannocke@msu.edu
Center: Michigan State University
Sample: Chromatin IP from total genomic DNA,H3K27me3 antibody. Flower bud tissue from Malus x domestica cv. Golden Delicious
Organism: Malus x domestica (cultivated apple) cv. Golden Delicious
Library: Apple_flower_GSLib-MID3
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: ChIP selected DNA was prepared for sequencing using the standard Roche/454 General Library construction protocol. In place of the standard 'A' primer a multiplex ID (MID, aka bar code) adapter was used. The MID used was the Roche/454 GSMID3 adapter. The MID tag sequence 'AGACGCACTC' immediately follows the key tag.
Platform: LS454
Instrument model: 454 GS FLX
Processing: none
Base calls: Base Space, 454 Basecaller
Quality score: 454 Basecaller, 64x1
18 Aug 2010 Chromatin-tagged sequence: M. x domestica cv. Golden Delicious open-pollinated seedling leaf [NCBI SRA Report SRX025561]
Experiment design: 454 sequencing of histone-modification-specific ChIP selected DNA.
Study Summary: Optimization of ChIP-seq for the characterization of expressed genes within bulk chromatin derived from apple (Malus x domestica)
Abstract: The goal of this study was to apply histone-modification-specific ChIP-seq for the identification of weakly expressed and tissue-specific expressed genes from total genomic DNA in domestic apple (Malus x domestica). We first determined if selected genes predicted to show strong tissue-specificity of expression could be recovered through ChIP using modification-specific antibodies. We targeted known sequences representing apple genes most closely related to Arabidopsis SVP, SOC1, TFL1, AP1, and AG. Using seedling tissues, we carried out extensive optimizations of crosslinking duration, sonication times and intensities, and antibody concentrations. Four of these sequences (SVP-like, SOC1-like, TFL1-like, and AP1-like) amplified selectively from the IP. An apple ACTIN sequence, which we know from EST source analysis to be expressed strongly in numerous tissues, was amplified only after extensive cycling, and did not show enrichment in the IPs. We then scaled up the ChIP using 10 g of seedling tissues and undertook a proof-of-concept experiment with ChIP-seq runs from apple flower bud and leaf. Of ~109,000 sequences from flower bud, ~21,000 matched 7,700 known apple contig/singleton sequences (BLASTN <1E-05), and ~8,800 matched ~4,000 Arabidopsis genes (TBLASTN <1E-05). Of ~226,000 sequences from leaf, ~72,100 matched ~14,300 known apple contig/singletons, and ~31,000 matched ~7,000 Arabidopsis genes. Given the abundance of unmatched, expressed-gene-like sequences in this dataset, we concluded that these limited preliminary experiments were highly successful in identifying previously unknown apple genes.
Contact Name: Steven van Nocker
Contact E-mail: vannocke@msu.edu
Center: Michigan State University
Sample: Chromatin IP from total genomic DNA,H3K27me3 antibody. Seedling leaftissue from open-pollinated Malus x domestica cv. Golden Delicious
Organism: Malus x domestica (cultivated apple) cv. Golden Delicious
Library: Apple_flower_GSLib-MID3
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: ChIP selected DNA was prepared for sequencing using the standard Roche/454 General Library construction protocol. In place of the standard 'A' primer a multiplex ID (MID, aka bar code) adapter was used. The MID used was the Roche/454 GSMID3 adapter. The MID tag sequence 'AGACGCACTC' immediately follows the key tag.
Platform: LS454
Instrument model: 454 GS FLX
Processing: none
Base calls: Base Space, 454 Basecaller
Quality score: 454 Basecaller, 64x1