ecotype: Col-0 tissue: mixture of ungrafted scions and stocks developmental stage: 22-26 hours after cut
Treatment protocol
4 days old seedlings were cut with razor blade to produce scion and stock. For making sample A, lift the scion up, bring it to the stock, and then pick up the stock carefully to approach the scion and connect them together. For sample B, the scions and stocks were not grafted and reserved until sampling at 22-26 hours after grafting. For sample C, intact seedlings were also reserved during grafting. Sample A, B and C were collected next day at 22-26 hours after grafting for RNA extraction.
Growth protocol
Arabidopsis thaliana ecotype Col-0 seeds were sown on the medium. The plates were incubated in the dark at 4°C for 2–3 days, then placed vertically in growth room (temperature: 22–25°C; light intensity: 6000 lux; photoperiod: 16h light, 8h dark) with the thin side pointing downward and the thick side upward. Grafting was performed using a dissecting microscope at 4 days post germination.
Extracted molecule
total RNA
Extraction protocol
Total RNA from frozen sample A, B and C were extracted by the Trizol reagent (Invitrogen) and purified with QIAGEN RNeasy® Mini Kit (QIAGEN) according to the manufacturer’s instructions. Total RNA concentration was assessed by spectrophotometry (OD 260 nm), and purity and integrity of the RNA were determined by the absorbance ratio at 260/280 nm and visualization after electrophoresis.
Label
Cy3
Label protocol
Total RNA was amplified and labeled by Low RNA Input Linear Amplification kit (Cat#5184-3523, Agilent technologies, Santa Clara, CA, US), 5-(3-aminoallyl)-UTP (Cat#AM8436, Ambion, Austin, TX,US), Cy3 NHS ester (Cat#PA13105,GE healthcare Biosciences, Pittsburgh, PA,US) followed the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
Each Slide was hybridized with 1.65μg Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US), according to the manufacturer’s instructions. After 17 hours hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit(Cat#5188-5327, Agilent technologies, Santa Clara, CA, US), Stabilization and Drying Solution (Cat#5185-5979, Agilent technologies, Santa Clara, CA, US)followed the manufacturer’s instructions.
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565BA, Agilent technologies, Santa Clara, CA, US) and Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US) with default settings，Scan resolution=5μm, PMT 100%，10%. Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
Description
Gene expression of ungrafted scions and stocks at 22-26hr after cut
Data processing
Data pre-processing and differential expression analysis of the gene expression data were done in R (version 2.10.0. Bioconductor, http://bioconductor.org/). Data were normalized between arrays using the quantile method. Normalized expression data was subjected to log2 transformation. For differential expression analysis, Student’s t test assumed that the variance of the two classes were not the same was applied. Firstly, data were analysed between A and C with the p-value ≤0.01, q-value≤0.05, and foldchange≥2. Differential probes were subsequently compared between A and B with p-value≤0.01, q-value≤0.05
