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assays/Maleckova2018_aba_mRNASeq/protocols/Illumina.md

+# Preparation of Illumina libraries, and sequencing
+
+Libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina) following the Illumina TruSeq Stranded mRNA Sample Preparation Guide #15031047 Rev.  E,  with  the  following  adaptations:  300  ng  total  RNA  as  the  basis  for library preparation, adapter index dilution to 50% with RNase-free water,  and  an  additional  purification  step  with  AMPure  XP  (Beckman  Coulter).The  concentration  of  the  resulting  libraries  was  estimated  using  a  NanoDrop  8000  (Thermo  Fisher  Scientific)  and  quality  control  was  performed  with  a  High  Sensitivity  NGS  Fragment  Analysis  Kit  (1  bp–6000 bp) DNF-474 (Advanced Analytical Technologies, Inc.). Based on  quantification  with  a  Qubit  dsDNA  HS  Assay  Kit  (Thermo  Fisher  Scientific), libraries were adjusted to 2 nM concentration before cluster generation with a HiSeq 3000/4000 SR Cluster Kit (Illumina). Twelve samples were pooled per lane and sequenced in 150 bp single-end mode on a HiSeq 3000 platform (Illumina). On average, over 32 million reads per library were obtained (Supplementary Table  S1).
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assays/Maleckova2018_aba_mRNASeq/protocols/RNAex.md

+# Total RNA extraction
+
+Total  RNA  was  extracted  from  ground  leaf  tissue  using  the  Monarch®Total  RNA  Miniprep  Kit  (New  England  Biolabs  Inc.)  following  the  manufacturer’s  instructions.  Contaminating  DNA  was  digested  with  DNase I (New England Biolabs Inc.). The integrity of input RNA was analysed on a 2100 Bioanalyzer (Agilent).
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studies/Maleckova2018_aba/README.md

+# Maleckova2018_aba
+
+Maleckova, E., Brilhaus, D., Wrobel, T.J., and Weber, A.P.M. (2019). Transcript and Metabolite Changes during the Early Phase of ABA-mediated Induction of CAM in Talinum triangulare. Journal of Experimental Botany 2: 16178–6596.
+
+https://doi.org/10.1093/jxb/erz189
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studies/Maleckova2018_aba/protocols/plant_material.md

+# Plant material and growth conditions
+
+Talinum  triangulare  plants  used  in  this  study  originated  from  two  sub-sequent,  controlled  self-pollination  events,  increasing  the  homogeneity  of the plant material. Seeds were germinated in multiplication substrate (Floraton 3, Floragard) and 4-week-old seedlings were transferred to pots with D 400 soil and Cocopor (Stender). Two weeks before the treatment, the  plants  were  placed  into  a  controlled-environment  plant  chamber  (MobyLux GroBanks, CLF Plant Climatics) with the following growth conditions: 12 h light/12 h dark at 25 °C/23 °C. The light intensity at the leaf level was 150–200 μmol s–1 m–2. To avoid unintended induction of  CAM  by  drought,  all  plants  were  watered  as  needed,  with  the  same  amount of water per pot.
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studies/Maleckova2018_aba/protocols/treatment_harvest.md

+# Treatments and harvest of leaf material
+
+To determine a suitable ABA concentration, a range from 10 to 600 μM was tested (see Supplementary Fig. S1 at JXB online) prior to the work presented here. Using 2-month-old plants, two mature and fully illumin-ated  leaves  of  each  plant  were  treated  either  with  (+/–)  ABA  [Sigma-Aldrich; 200 μM solution in 0.095% (v/v) methanol] or mock solution (0.095% (v/v) methanol). Both solutions contained 0.02% (v/v) Tween-20 (PanReac AppliChem). The first treatment took place after 4 hours in the light and the second treatment followed 4 hours later (Fig. 1). Treated leaves were harvested and snap-frozen in liquid nitrogen 40, 80, 160, 320, 640,  and  1280  min  after  the  first  treatment.  Two  biological  replicates  originating from two independent plants were harvested per time point. The treated leaves from the same plant were pooled prior to all analyses.
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