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| Status |
Public on Jan 25, 2017 |
| Title |
hdac_K14 |
| Sample type |
SRA |
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| Source name |
Young seedlings
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| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: Seedling
developmental stage: 10-day-old
antibody: H3K14ac (Abcam, ab52946)
genotype/variation: hda9-1
ecotype: Col
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| Growth protocol |
Seeds were surface-sterilized and sown on MS-agar plates. After two days of cold treatment, the plated seeds were transferred to 23°C under long-day (16 h light, 8 h dark) condition.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Seedlings were ground in liquid nitrogen and cross-linked with 1% formaldehyde in M1 buffer [10 mM phosphate buffer pH 7.0, 0.1 M NaCl, 10 mM mercaptoethanol, 1 M hexylene glycol, protease inhibitor cocktail (Roche)] for 10 min. The cross-linking reaction was quenched by adding glycine. The suspension was filtered through four layers of Miracloth, and the filtrate was centrifuged at 12,000 rpm for 10 min. The pelleted chromatin was washed three times with M2 buffer (M1 buffer plus 10 mM MgCl2 and 0.5% Triton X-100) and one time with M3 buffer [10 mM phosphate buffer pH 7.0, 0.1 M NaCl, 10 mM mercaptoethanol, protease inhibitor cocktail (Roche)]. The chromatin was resuspended in nuclei lysis buffer and sonicated to generate DNA fragments approximately 200bp in length. The lysate was pre-cleared by incubation with 50 µl protein-A agarose beads/salmon sperm DNA (Millipore) for 1 h then incubated with anti-H3K9ac (Abcam, ab10812) and anti-H3K14ac antibodies (Abcam, ab52946) overnight. The beads were washed two times with each of the following solutions: low salt, high salt, LiCl and TE. The bound DNA fragments were recovered and purified using columns from the Qiagen Plasmid Extraction Kit according to the manufacturer’s instructions.
ChIP DNA was quantified using a fluorescence-based quantification method (Qubit, Invitrogen). ChIP-seq libraries were prepared according to the manufacturer’s instructions (Illumina TruSeq ChIP sample prep kit). Input ChIP DNA was blunt-ended and phosphorylated, and a single adenine base was added to the 3' ends of the fragments in preparation for ligation to an adapter with a single thymine base overhang. The products of this ligation reaction were purified and size-selected (300 to 400 bp fragments) by 2% agarose gel electrophoresis. The size-selected DNA was purified and PCR-amplified to enrich for fragments with adapters on both ends. The quality of the amplified libraries was verified by capillary electrophoresis (Bioanalyzer, Agilent). After qPCR using SYBR Green PCR Master Mix (Applied Biosystems), the index-tagged libraries were combined in equimolar amounts within a single pool. Sequencing was performed using an Illumina NextSeq 500 system following the provided protocols for 1x75 sequencing.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
hdac_K14
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| Data processing |
Basecalls and quality control and pre-aligned (eland) were performed using Illumina Genome analyzer pipeline
ChIP-seq reads were aligned to the Arabidopsis genome (TAIR10) using BOWTIE.
Perfect and unique reads from the mapping were processed for further analysis. Wiggle (WIG) files of the alignments were generated using bam2wig and visualized using IGV. Using the SICER program, ChIP-enriched peaks were identified, and qualitative comparisons of binding levels in wild-type and mutant plants were determined.
Genome_build: TAIR10
Supplementary_files_format_and_content: peaks bed files
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| Submission date |
Nov 10, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Lei Gao |
| E-mail(s) |
leigao@szu.edu.cn
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| Organization name |
shenzhen university
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| Department |
College of Life Sciences
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| Street address |
Nanhai Ave 3688
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| City |
shenzhen |
| State/province |
guangdong |
| ZIP/Postal code |
518060 |
| Country |
China |
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| Platform ID |
GPL17639 |
| Series (2) |
| GSE89768 |
POWERDERESS and HDA9 interact and promote histone H3 deacetylation at specific lysine residues in Arabidopsis. [ChIP-Seq] |
| GSE89770 |
POWERDERESS and HDA9 interact and promote histone H3 deacetylation at specific lysine residues in Arabidopsis. |
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| Relations |
| BioSample |
SAMN06010019 |
| SRA |
SRX2342089 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2388442_hdacK14.peaks.bed.gz |
99.7 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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