GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1862908

Query DataSets for GSM1862908

Status

Public on Aug 28, 2015

Title

Col-0 16h

Sample type

SRA

Source name

Infected Arabidopsis thaliana leaf tissue

Organism

Arabidopsis thaliana

Characteristics

ecotype: Col-0
genotype/variation: wild type
agent: Pseudomonas syringae pv. tomato DC3000

Treatment protocol

Leaf infiltration: Plants were randomised and for each genotype and time-point 3 fully expanded leaves from four individual plants were either harvested (non-induced) or inoculated with DC3000. For infiltration, a small nick was made on either side of central vascular vein on the abaxial surface and bacterial challenge (OD600 of 0.15) was performed with a 1ml needleless syringe. All inoculations were completed by 11 am and inoculated plants were left under a light bench in the laboratory (22oC). Samples were taken at 6, 8, 12 and 16 hpi. Leaves were harvested by cutting at the petiole/leaf blade junction and were immediately snap-frozen in foil untill used for RNA preparation.

Growth protocol

Arabidopsis Sowing And Plant Growth Conditions: Arabidopsis thaliana seed, Col-0 or jaz5/10, were sown onto ~7cm square pots of sieved compost mix (Levingtons F2 compost+sand (LEV206): vermiculite (medium grade) mixed in a 6:1 ratio) and stratified for 3 days at 4°C in darkness. Seeds were germinated in a controlled environment growth chamber with a 10 h photoperiod (lights on 8am, off 6pm) of 120 µmol m-2 s- at 23oC (day), 20oC (night) and relative humidity of 65%. Plants were pricked into individual 7cm pots in a 24 cell and plants were grown for 4.5 weeks. Cells were continuously moved in the controlled environment chamber to reduce positional effects. Pseudomonas growth: Pseudomonas syringae pv. tomato strain DC3000 carrying the empty broad host range vector pVSP61 (Innes et al., 1993) was grown on solidified Kings B media (King et al. 1954) containing rifampicin 50 μg ml-1 and kanamycin 25 μg ml-1. For inoculation, overnight cultures were grown with shaking (200 rpm) at 28oC. Cells were harvested (2000g X 8 min), washed and resuspended in 10 mM MgCl2. Cell density was adjusted to OD600 0.15 ( ~0.75 x 108 colony forming units (cfu) ml-1).

Extracted molecule

total RNA

Extraction protocol

Plant RNA extraction: RNA was extracted according to de Torres et al. (2006). The frozen single leaf was ground to a fine powder in a liquid nitrogen pre-cooled mortar. 600µl of Z6 buffer (8M guanidinium hydrochloride, 20mM MES, 20mM EDTA pH7.0) was added and the powder quickly mixed. When thawed, the sample was passed to a 1.5ml microfuge tube and the mortar washed with 100µl of Z6 buffer. The combined sample was extracted with 1 vol. of phenol/chloroform then centrifuged at 4oC for 5 min at maximum speed. The aqueous phase was removed to a new microfuge tube and 1/20 vol. of acetic acid (1M) and 0.7 vol. 100% ethanol added nucleic acids collected after 30min on ice. The resultant RNA pellet was washed in 70% ethanol and resuspended in 100µl of sterile ddH2O. Samples were cleaned up using a Qiagen RNeasy Plant mini kit according to the manufacturers instructions and samples eluted in 30μl in RNAse free water.
2 µl of diluted ERCC RNA control spike-in mix 1 or control spike-in mix 2 (Life Technologies) was added to 1 µg of sample RNA and the volume adjusted to 50 µl. Sequencing Libraries were prepared usingthe standard Illumina TruSeq RNA library preparation protocol. on a Sciclone NGS platform. Samples were processed in 96 well hard shell PCR plates (Biorad HSP-9631). Washes were done in 150 µl bead washing solution instead of the 200 µl used in the manual process. Incubations at 65°C and 16°C for mRNA denaturation, and second-strand synthesis were performed on the Sciclone deck. However, the 80°C and 94°C incubations for mRNA elution and fragmentation, and first-strand cDNA synthesis reactions were performed on a thermocycler with heated lid to avoid excess evaporation. Purification and size selection was by Ampure XP bead clean up rather than gel based size selection. Library quality and quantitiy was determined by LabChipGX high sensitivity assay, after diluting the libraries 2µl in 20 µl.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer II

Data processing

Base Calling carried out using CASAVA v1.8.2
Fastq-Mcf was then used to remove adapter sequences and trim reads of a phred quality score < 20 with the following parameters: -q 20 -l 35 -k 0 -p 15 --max-ns 0 -m 3. ERCC reads were removed using Bowtie v1.0.0 with the following parameters: -S -X 600
Reads were aligned to the reference genomes for Arabidopsis thaliana using TopHat v2.0.8b with the following parameters: -r 200 --mate-std-dev 375 --library-type fr-unstranded.
Reads were quantified at each predicted gene location using htseq-count v0.6.0 with the following parameters: -m union -s yes
These single replicate data were analysed by gFOLD (Feng et al., 2012) using a cutoff of log2 1.6
Supplementary_files_format_and_content: Tab delimited matrix containing raw counts, gene length and the normalised count for each gene in each sample.

Submission date

Aug 27, 2015

Last update date

May 15, 2019

Contact name

ZhengRong Yang

E-mail(s)

z.r.yang@ex.ac.uk

Phone

01392 263448

Organization name

Exeter University