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SRX1044959: GSM1701081: ChIP-Seq analysis of TRB1 associated sequences_(TRB1(+)_anti-TRB1); Arabidopsis thaliana; ChIP-Seq
2 ILLUMINA (Illumina HiSeq 2500) runs: 39.7M spots, 2G bases, 1.2Gb downloads

Submitted by: Gene Expression Omnibus (GEO)
Study: Telomere Binding Protein TRB1 Preferentially Associates with Promoters of Translation Machinery genes
show Abstracthide Abstract
Background: Recently we characterised TRB1, a protein from a single-myb-histone family, as a structural and functional component of telomeres in Arabidopsis thaliana. TRB proteins, besides their ability to bind specifically telomeric DNA using their N-terminally positioned myb-like domain of the same type as in human TRF1 or TRF2 shelterin proteins, possess also histone-like domain which is involved in protein-protein interactions e.g., with POT1b. We set to investigate the genome-wide localization pattern of TRB1 to reveal its preferential mode of binding to chromatin in vivo and its potential functional roles in the genome-wide context. Results: Our results demonstrate that in addition to its roles at telomeres, TRB1 is preferentially associated with promoter regions of genes involved in ribosome biogenesis. This preference coincides with a frequent occurrence of telobox motifs in the upstream regions of genes in this category, but is not restricted to the telobox presence. Conclusions: We conclude that TRB1, in addition to its preferential telomeric localization, shows a specific genome-wide distribution pattern suggesting its role in regulation of genes involved in biogenesis of translational machinery. Overall design: TRB1 DNA-binding activity investigated in 2 biological replicates (2 immunoprecipitation approaches) with controls; with technical replicates.
Sample: ChIP-Seq analysis of TRB1 associated sequences_(TRB1(+)_anti-TRB1)
SAMN03754341 • SRS950312 • All experiments • All runs
Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Selection: ChIP
Layout: SINGLE
Construction protocol: The ChIP assay was performed as described Bowler et al. (2004) with modifications. Chromatin extracts were prepared from seedlings treated by 1% formaldehyde. The chromatin from isolated nuclei was sheared to an average length of 250-500 bp by sonication (Bioruptor, Diagenode) and spun. Matrix GFP-TRAP_A (Chromtec) was blocked against non-specific interaction with 200 mM ethanolamine, 1% BSA and TR10-24-G and TR10-24-C (i.e. DNA sequences, that are not recognised by TRB proteins (Schrumpfova et al., 2004). The pretreated matrix was incubated with chromatin diluted with ChIP dilution buffer (16.7 mM Tris-HCl, pH=8,0, 1.2 mM EDTA, 167 mM NaCl, 0.1% TritonX-100, PMSF and protease inhibitors) for 4°C / 4h and consequently washed with low salt; high salt; LiCl; TE buffers (in contrast to Bowler et al. (2004) levels of detergents (TritonX-100, NP-40 and sodium deoxycholate) were lowered to 0.1%). The cross-linking was reversed by 0.2 M NaCl O/N and followed by proteinase K (Serva) treatment, phenol/chlorophorm extraction and RNase A (Serva) treatment according to Bowler et al. (2004). Fifty microliters of immunoprecipitated DNA (ranging from 0.2-6 ng DNA (measured by Qubit dsDNA HS Assay Kit (Invitrogen)) was used for library preparation using NEBNext ChIP Seq Liabrary Prep Master Mix (NEB), with Agencourt XP beads (Beckman) in the ratios described in the protocol only with one exception: the Adapter was diluted 1:17 in water. DNA fragments were selected by 2% e-gel (Invitrogen) in the 270-300bp size range. Subsequently, a PCR reaction (18 cycles) with indexed Primers 1-11 from NEBNext Multiplex Oligo set (NEB) was performed. Libraries were sequenced (50 bp single-end reads) on an Illumina HiSeq 2500 using TruSeq SBS Kit v3-HS (Illumina) sequencing reagents. The quality of the final libraries was checked on the Bioanalyzer (Agilent) and the quantity was checked with Qubit dsDNA HS Assay Kit (Invitrogen). The libraries were pooled equimolar and diluted to 10 pM (denaturation in NaOH) in Hyb Buffer 1 (HT1) from TruSeq SR Cluster Kit v3- cBot HS (Illumina).
Experiment attributes:
GEO Accession: GSM1701081
External link:
Runs: 2 runs, 39.7M spots, 2G bases, 1.2Gb
Run# of Spots# of BasesSizePublished


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