Water + Water rep A
Arabidopsis thaliana
Protocols: Seedlings were collected with forceps and blotted dry on tissue paper. Seedlings were placed in safelock 1.5ml eppendorf tubes and immediately frozen in liquid nitrogen before storing at -80 degrees celcius. Seeds were sterilised for 15 min. with 50% bleach, 0.1% triton and thoroughly washed with sterile water. Seeds were stratified at 4 degrees celsius for 2-3 days and then were germinated on vertical standing MS plates. 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 10ml of water or 1mM NHP for 18 hours. Seedlings were then removed, blotted dry with tissue paper, then transferred to another 6 well plate and immersed in 10ml water or 0.5mM SA for 6 hours. 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). 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 Beijing Genomics Institute (BGI), the protocol of which is proprietary.
ENA first public
2025-03-01
INSDC center name
The University of Edinburgh
INSDC status
public
Submitter Id
E-MTAB-14847:Water + Water rep A
age
12
broker name
ArrayExpress
collection date
not collected
common name
thale cress
compound 2
water
developmental stage
seedling development stage
genotype
wrky38/wrky62 mutant
geographic location (country and/or sea)
not collected
growth condition
long day length regimen
isolate
not applicable
organism part
whole organism
sample name
E-MTAB-14847:Water + Water rep A
scientific_name
Arabidopsis thaliana
ecotype
Col-0