description: Protocols: Seedlings were collected and placed into a 2 ml screw-cap tube and immediately frozen in liquid nitrogen before storing at -80 degrees celsius. Seeds were sterilised for 5 min. with 70% ethanol followed by 5 min. with 10% bleach and thoroughly washed with sterile water. Seeds 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 floated on 10 ml of water or 50 uM ACC. Samples were collected at 3 hours after treatment. 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 Beijing Genomics Institute, 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: SAMEA10159277
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-10964:upl3 upl4 ACC rep_b
attributes.tag: age
attributes.value: 10
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: thale cress
attributes.tag: developmental stage
attributes.value: seedling development stage
attributes.tag: ecotype
attributes.value: Col-0
attributes.tag: genotype
attributes.value: upl3 (SALK_ 035524) upl4 (SALK_040984)
attributes.tag: organism part
attributes.value: whole organism
attributes.tag: sample name
attributes.value: E-MTAB-10964:upl3 upl4 ACC rep_b