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Sample GSM2388441 Query DataSets for GSM2388441
Status Public on Jan 25, 2017
Title hdac_input
Sample type SRA
 
Source name Young seedlings
Organism Arabidopsis thaliana
Characteristics tissue: Seedling
developmental stage: 10-day-old
genotype/variation: hda9-1
ecotype: Col
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.
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.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
 
Description hdac_input
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
 
Submission date Nov 10, 2016
Last update date May 15, 2019
Contact name Lei Gao
E-mail(s) leigao@szu.edu.cn
Organization name shenzhen university
Department College of Life Sciences
Street address Nanhai Ave 3688
City shenzhen
State/province guangdong
ZIP/Postal code 518060
Country China
 
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.
Relations
BioSample SAMN06010017
SRA SRX2342088

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not applicable for this record

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