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Sample GSM2665533

Query DataSets for GSM2665533

Status

Public on Jun 13, 2017

Title

col0 - mock treated replicate2

Sample type

SRA

Source name

Arabidopsis thaliana 14-day-old in vitro seedlings

Organism

Arabidopsis thaliana

Characteristics

ecotype: Col-0
genotype: Wt
treatment: none
tissue: seedlings
age: 14-day old

Treatment protocol

For flagellin treatments (flg treated), seeds were surface-sterilized by treatment with bayrochlore and then soiled in sterile half-strenght MS liquid medium, placed for 2-4 days at 4°C to obtain homogeneous germination, and plants were grown in chambers at 20°C in long-days (16h of light) conditions. After 14 days, flg22 peptide was added in the medium to a final concentration of 1μm when necessary. For the mock condition (mock treated), the same volume of water was added to the medium instead of the flg22 peptide.

Growth protocol

14-day-old in vitro seedlings were used. T-DNA insertion lines hd2b (At5g22650) from SAIL collection 41 Sail_1247_A02 and mpk3 SALK_151594 42 were obtained from the the Nottingham Arabidopsis Stock Centre (NASC).

Extracted molecule

total RNA

Extraction protocol

[ChIP-seq] ChIP assays were performed on 14-day-old in vitro seedlings using anti-GFP , IgG control, or anti-H3K9ac antibodies. Briefly, after plant material fixation in 1% (v/v) formaldehyde, tissues were homogenized, nuclei isolated and lysed. Cross-linked chromatin was sonicated using a water bath Bioruptor UCD-200 (Diagenode, Liège, Belgium) (30 s on/30 s off pulses, at high intensity for 60 min). Protein/DNA complexes were immunoprecipitated with antibodies, overnight at 4°C with gentle shaking, and incubated for 1h at 4°C with 50 μL of Dynabeads Protein A (Invitrogen, Ref. 100-02D). Immunoprecipitated DNA was then recovered using the IPure kit (Diagenode, Liège, Belgium) and analyzed by quantitative real-time PCR. An aliquot of untreated sonicated chromatin was processed in parallel and used as the total input DNA control.
[ChIP-seq] 10 ng of IP or input DNA was used for ChIP-Seq library construction using NEB-Next Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer’s recommendations. For all libraries, twelve cycles of PCR were used. The quality of the libraries was assessed with Agilent 2100 Bioanalyzer (Agilent), and the libraries were subjected to high-throughput sequencing by Illumina Sequencing technology.
[RNA-seq] Total RNA were extracted from seedlings with the RNeasy MiniPrep kit (Qiagen), according to the manufacturer's instructions.
[RNA-seq] Transcriptomic analyses were performed RNA-seq at the URGV plateform (Evry, France) with Illumina RNA-seq preparation kit. Three biological replicates were run for each condition.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

HD2B_RPKM_RNAseq.txt

Data processing

The reads were mapped onto Arabidopsis thaliana TAIR10 genome
To identify regions that were significantly enriched, we used MACS2.
MACS2 parameters for peaks detection were set as follows: Number of duplicate reads at a location:1; Bandwidth:300; mfold of 5:50; q-value cutoff:0.05.
To identify the differentially expressed genes, we used UGRV platform
Genome_build: TAIR10
Supplementary_files_format_and_content: [ChIP-Seq] wig files report peaks
Supplementary_files_format_and_content: [RNA-Seq] txt file reports RPKM values

Submission date

Jun 12, 2017

Last update date

May 15, 2019

Contact name

Alaguraj Veluchamy

E-mail(s)

alaguraj.v@gmail.com

Organization name

King Abdullah University of Science and Technology

Department

BESE