Illumina HiSeq 2500 sequencing; Salicylic acid-induced gene expression in wild-type Col-0 and mutant ube4 Arabidopsis thaliana plants
Salicylic acid-induced gene expression in wild-type Col-0 and mutant ube4 Arabidopsis thaliana plants
Seedlings were collected with forceps and blotted dry on paper tissue. Subsequently seedlings were placed into a 2 ml screw-cap tube and immediately frozen in liquid nitrogen before storing at -80 degrees celcius.  Seeds were sterilised for 5 min. with 70% ethanol followed by 5 min. with 10% bleach and thoroughly washed with sterile water. Seeds were then resuspended in sterile 0.5% agar. Seeds were germinated on vertical standing MS plates supplemented with 1X Gamborg vitamins. Growth conditions: 16/8 hours light/dark cycle with 120 mol m-2 s-1 light and 22 degrees Celsius.  Seedlings were placed in 6-well plates and immersed in 10 ml of water or 0.5 mM SA.  1) Grind tissue very fine in liquid nitrogen. 2) Add 0.5 ml RNA extraction buffer (warmed to ~80ºC) and 0.5 ml phenol:chloroform:isoamylalcohol mixture (25:24:1). Vortex vigorously. 3) Centrifuge max. speed for 5 min. at 4ºC, but do not put samples on ice. 4) Transfer the aqueous phase (upper phase) to a new tube. 5) Add 0.5 ml cold chloroform:isoamylalcohol (24:1) and vortex vigorously. 6) Centrifuge max. speed for 5 min. at 4ºC, but do not put samples on ice. 7) Repeat steps 4, 5, and 6. 8)Transfer the aqueous phase to a new tube on ice and add 1/3 volume of 8 M LiCl.  9) Incubate overnight at 4ºC. 10) Centrifuge max. speed for 15 min. at 4ºC. A small clear pellet of total RNA should  be visible. From now on keep tubes on ice at all times. 11) Without disturbing the pellet, very carefully remove the supernatant with a pipette. 12) Wash RNA pellet gently with ice-cold (-20ºC) 0.5 ml of 70% ethanol. Do not resuspend pellet. 13) Centrifuge max. speed for 1 min. at 4ºC to make sure the RNA pellet stays in place.Discard supernatant without disturbing the RNA pellet. 14) Repeat step 13 to remove any remaining 70% ethanol. 15) Dry RNA pellet in the SpeedVac for 1 min. without heating. Do not dry longer than this!!! 16) Add 400 μl ddH2O (autoclaved) and keep on ice for 30-60 min. to rehydrate the RNA pellet. 17) Dissolve the RNA pellet by vigorously pipetting the solution up and down.  18) Add 40 μl NaAc pH5.2 and 1 ml of ice-cold (-20ºC) 96% ethanol. Invert to mix (do not vortex!!!). 19) Incubate at least 60 min. at -20ºC.  20) Centrifuge max. speed for 15 min. at 4ºC. A gel-like pellet of pure RNA should now be visible. 21) Without disturbing the pellet, very carefully remove the supernatant with a pipette. 22) Wash RNA pellet gently with ice-cold (-20ºC) 0.5 ml of 70% ethanol. Do not resuspend pellet!!! 23) Centrifuge max. speed for 1 min. at 4ºC to make sure the RNA pellet stays in place. Discard supernatant without disturbing the RNA pellet. 24) Repeat step 23 to remove any remaining 70% ethanol. 25) Dry RNA pellet in the SpeedVac for 1 min. without heating. Do not dry longer than this! 26) Add 25 μl ddH2O (autoclaved) and let sit on ice for 30 min. to rehydrate the RNA pellet. 27) Resuspend RNA pellet by pipetting up and down. 28) RNA was then purified further using an RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions.  Buffers & Solutions:RNA extraction buffer (autoclave): 100 mM Tris-HCl, pH 8.0;  100 mM LiCl; 10 mM EDTA; 1% SDS; Phenol:chloroform:isoamyl alcohol (25:24:1); Chloroform:isoamylalcohol (24:1); 8M LiCl (autoclave); 3M NaAC, pH 5.2 (autoclave;)70% Ethanol; 96% Ethanol (pure ethanol) Libraries were constructed by GATC/Eurofins, the protocol of which is proprietary.
Experimental Factor: genotype
npr1-1
Experimental Factor: compound
none