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| Status |
Public on Sep 29, 2017 |
| Title |
HTA11_ChIP-seq_27c_15min_rep3 |
| Sample type |
SRA |
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| Source name |
Col0 HTA11::HTA11:FLAG seedlings
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| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype: HTA11::HTA11:FLAG
cultivar: Col0
tissue: seedling
shift experiment type (17>27c or 17c>37c): 17c>27c
temperature: 27c
incubation time: 15min
replicate: 3rd
fragmentation method (mnase digestion or sonication): MNase (mononucleosome)
chip antibody: Anti-FLAG® M2 Magnetic Beads (M8823 SIGMA)
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| Growth protocol |
Seedlings were grown at 17c in long days for 12 days and collected after 15min, 1hr or 4hrs of shift 17c, 27c or 37c.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed as described (Gendrel et al., 2002) with minor modifications. In brief, chromatin was extracted from 1 g of crosslinked materials. For HTA11 (a highly expressed H2A.Z gene) and H3, the chromatin was resuspended in MNase digestion buffer (20mM Tris-HCl [pH8], 50mM NaCl, 1mM DTT, 0.5% NP-40, 1mM CaCl2, 0.5mM phenylmethylsulfonyl fluoride (PMSF) and 1X protease inhibitor cocktail [Roche]), and digested with 0.4U/ml of Micrococcal nuclease (MNase, Sigma, N3755) for 15 min. Digestion was stopped with 5 mM EDTA. For HSF1, chromatin was fragmented by sonication using a bioruptor (Diagenode) in lysis buffer (10 mM Tris-HCl [pH 8], 150 mM NaCl, 1 mM EDTA [pH 8], 0.1% deoxycholate, and 1X protease inhibitor cocktail). All ChIPs were performed in a buffer containing 20mM Tris-HCl (pH8), 150mM NaCl, 2mM EDTA, 1% triton X-100 and 1X protease inhibitor cocktail. ChIP were performed for HTA11 and HSF1 using FLAG M2 magnetic beads (Sigma, M8823) and for H3 using H3 antibody (Abcam, ab1791) coupled to a 1/1 mix of protein-A and protein-G Dynabeads (life technologies, 10001D and 10003D). All ChIPs were performed in a buffer containing 10 mM Tris-HCl [pH 8.0], 5 mM EDTA [pH 8.0], 150 mM NaCl, 1% Triton X-100, and 1× protease inhibitor cocktail. The elution was done for HTA11 and HSF1 ChIP with 100ng/ul of 3XFLAG in TE buffer and for H3 ChIP with elution buffer (1% SDS, 0.1M NaHCO3).
The efficiency of ChIP materials was assessed by qPCR in prior to in-house library preparation, using the TruSeq ChIP sample preparation kit (Illumina, IP-202-1012) following the manufacturer’s instructions. The libraries were sequenced on HiSeq2000 by MPI Tubingen and BGI using paired-end 100bp length reads, and on NextSeq500 at the Cambridge Genomic Services sequencing facility, Department of Pathology, University of Cambridge and at the Sainsbury Laboratory Cambridge University using paired-end 75 bp length reads .
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
The raw reads obtained from the sequencing facilities were analysed in house. We first assessed the quality of reads using FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/). Potential adaptor contamination and low quality trailing sequences were removed using Trimmomatic (Bolger et al., 2014), before being aligned to the TAIR10 reference genome using Bowtie2 (Langmead et al., 2009). Potential optical duplicates were removed using Picard, as described earlier. The read counts mapped to each bp in each sample was normalised by the sample’s genome-wide mappable reads coverage per bp, and used in the subsequent statistical analyses. The nucleosome and ChIP profiles were binned to generate a “pileup” ChIP profiles for different groups of genes/promoters using in-house R and Perl scripts. Nucleosome positioning and occupancy was determined using DANPOS (Chen et al., 2013). HSF1 peaks were analysed using MACS (Zhang et al., 2008) and PeakAnalyzer (Salmon-Divon et al., 2010).
The nucleosome and ChIP profiles were binned to generate “pileup” ChIP profiles at TSS using in-house R and Perl scripts. We restricted the analyse to the genes of 750 bp or longer in length, and with at least 500 bp upstream intergenic region between the adjacent genes, in order to avoid false gene body H2A.Z signal influenced by H2A.Z that marks the start or stop of the adjacent genes.
Genome_build: TAIR10
Supplementary_files_format_and_content: text files with binned reads at TSS.
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| Submission date |
Mar 17, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Sandra Cortijo |
| E-mail(s) |
sandra.cortijo@slcu.cam.ac.uk
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| Organization name |
Sainsbury Laboratory, Cambridge University
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| Street address |
Bateman Street
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| City |
Cambridge |
| ZIP/Postal code |
CB2 1LR |
| Country |
United Kingdom |
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| Platform ID |
GPL19580 |
| Series (2) |
| GSE79354 |
Genome-wide analysis of transcription, H2A.Z, nucleosomes and HSF1 dynamics in response to temperature increase in Arabidopsis thaliana [ChIP-seq & Mnase-seq] |
| GSE79355 |
Genome-wide analysis of transcription, H2A.Z, nucleosomes and HSF1 dynamics in response to temperature increase in Arabidopsis thaliana |
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| Relations |
| BioSample |
SAMN04565980 |
| SRA |
SRX1640035 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2092780_TSS_V10_all_gene_TAIR10_27d15min_hta11_rep3_500_500_10_0_750.txt.gz |
6.4 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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