title: Illumina HiSeq 2500 paired end sequencing; Salicylic acid-induced gene expression in wild-type Col-0 and mutant upl3-4 Arabidopsis thaliana plants.
design: Salicylic acid-induced gene expression in wild-type Col-0 and mutant upl3-4 Arabidopsis thaliana plants.
library_construction_protocol: Tissue was collected by excising leaves at the petiole. Leaves were then packaged in aluminium foil and frozen in liquid nitrogen before storing at -80 degrees celcius.  Seeds were germinated on soil in 100% relative humidity. After 12 days seedlings were transplanted to larger pots (6 plants per pot) and grown for an additional 3 weeks. Plants were watered as required. Growth conditions: 16/8 hours light/dark cycle (120 mol m-2 s-1), 21/18 degrees Celsius day/night cycle, 65% relative humidity.  Plants were sprayed with a spray bottle that produced fine mist. Leaves were sprayed with water or 0.5 mM SA until thoroughly covered in fine droplets. After 24 hours leaf tissue was harvested for analysis.  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. Optional: If protocol was started with small amounts of tissue also add 25 µg glycogen (5 µl of 5 mg/ml stock) to visualize RNA pellet in step 10. 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) consists of 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) Strand-specific RNA libraries were constructed by GATC/Eurofins, the protocol of which is proprietary.
attributes.tag: Experimental Factor: compound
attributes.value: none
attributes.tag: Experimental Factor: genotype
attributes.value: wild type genotype