description: Protocols: Leaves were collected and placed into a 2 ml screw-cap tube and immediately frozen in liquid nitrogen before storing at -80 degrees celcius. Seeds were germinated on soil in 100% relative humidity. After 10 days seedlings were transplanted to larger pots (6 plants per pot) and grown for an additional 18 days. 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: ENA first public
attributes.value: 2021-10-06
attributes.tag: ENA last update
attributes.value: 2021-10-06
attributes.tag: External Id
attributes.value: SAMEA10159129
attributes.tag: INSDC center alias
attributes.value: Institute of Molecular Plant Sciences School of Biological Sciences University of Edinburgh
attributes.tag: INSDC center name
attributes.value: Institute of Molecular Plant Sciences School of Biological Sciences University of Edinburgh
attributes.tag: INSDC first public
attributes.value: 2021-10-06T00:47:56Z
attributes.tag: INSDC last update
attributes.value: 2021-10-06T00:47:56Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10963:upl5 SA rep_a
attributes.tag: age
attributes.value: 28
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: thale cress
attributes.tag: ecotype
attributes.value: Col-0
attributes.tag: genotype
attributes.value: upl5 (SALK_116446)
attributes.tag: organism part
attributes.value: leaf
attributes.tag: sample name
attributes.value: E-MTAB-10963:upl5 SA rep_a