tissue: stem base genotype: cv Taichung 65, crl1 mutant line: DXC hour after induction: 6
Treatment protocol
Plantlets were treated by adding dexamethasone (DEX) (ref. D4902, Sigma-Aldrich, MO, USA) diluted in absolute ethanol, to obtain a final concentration of 1 µM. After 5 hour, DEX was removed by replacing the culture medium with a fresh one.
Growth protocol
Oryza sativa L. cv. Taichung 65 crl1 mutant (Inukai et al. 2005) was transformed with a binary vector pINDEX2 (Ouwerkerk et al., 2001, Planta 213, 370-378) empty or containing the CRL1 gene (Os03g0149100) to generate DX and DXC lines respectively as described in Coudert et al., 2015 New Phytol 206(1):243-54. The line DXC corresponds to line DXC3 in Coudert et al., 2015, 206(1):243-54). Seeds were dehulled and disinfected (3 min in ethanol 70%, 90 min in 2:3 v/v sodium hypochlorite 9,6%/Milli-Q water plus 500 µL Tween 80, quadruple rinsed in Milli-Q water) and inoculated under sterile conditions in round Petri dishes (ref. 82.1473.001, Starstedt, Germany) containing a filter pad (ref. 1001-090, Whatman paper, GE Healthcare, UK) and 15 mL half strength Murashige and Skoog (MS/2) medium (ref. M0231, Duchefa Biochemie, The Netherlands). Culture chambers were set at 27°C, 70% relative humidity, and 14 hour daylight. After 5 days, the plantlets were transferred into 250 mL wide-collar Erlenmeyer flasks containing 30 mL MS/2 medium, and let to rest for 24 hour.
Extracted molecule
total RNA
Extraction protocol
Stem bases were collected every 3 hour from 0 to 45 hour after adition of dexamethasone in the medium (hours after induction, hai), according to Coudert et al., 2011, BMC Genomics, 12, e387. The experiment was repeated independently three times with DX and DXC lines. For each biological replicate, ten to fifteen stem bases were collected and immediately frozen in liquid nitrogen. After grounding 8~12 stem bases in liquid nitrogen using a TissueLyser II tissue disruption system (Qiagen, The Netherlands) with 3 mm steel beads for 15 sec at 30 Hz, RNA were extracted using the Plant RNeasy Kit (Qiagen, The Netherlands) with DNAse I treatment (Qiagen) on purification column according to the supplier’s recommendations.
Label
biotin
Label protocol
Biotinylated single strand cDNA were prepared according to the Affymetrix WT PLUS protocol from 200 ng total RNA (WT PLUSTechnical Manual, 2010 - 2012, P/N 702935 Rev4 Affymetrix).
Hybridization protocol
Following fragmentation and biotin end labeling, 3,8 ug of cDNA were hybridized for 20h at 48C° on Affymetrix® Rice Gene 1.1ST Jp array. GeneChips were washed and stained in the Affymetrix Fluidics Station 450 of Affymetrix GeneAtlas system using Hybridization, Wash & Stain kit.
Scan protocol
GeneChips were scanned using the Affymetrix GeneAtlas Scanner.
Description
Gene expression data from stem base of DXC line 6 h after dexamethasone treatment
Data processing
Data were generated with Affymetrix Epression Console v 1.4.1 and GCRMA algorithm.
Time-series transcript profiling analysis in stem base of rice crown rootless 1 mutant after ectopic expression induction by dexamethasone of the CRL1 gene
