diff --git a/assays/CMML_18-0008_GC-MS/isa.assay.xlsx b/assays/CMML_18-0008_GC-MS/isa.assay.xlsx index ddb29d440999a2b2c1a628e4adbe169d027a6df5..aef434cea1039931d53f11c4a3dec99b8df8644f 100644 Binary files a/assays/CMML_18-0008_GC-MS/isa.assay.xlsx and b/assays/CMML_18-0008_GC-MS/isa.assay.xlsx differ diff --git a/assays/CMML_18-0008_GC-MS/protocols/GC-MS.md b/assays/CMML_18-0008_GC-MS/protocols/GC-MS.md new file mode 100644 index 0000000000000000000000000000000000000000..6f481cbd427cfc529a0480103732edb15cee80d0 --- /dev/null +++ b/assays/CMML_18-0008_GC-MS/protocols/GC-MS.md @@ -0,0 +1,3 @@ +## Metabolite profiling + +Approximately 50 mg of ground leaf material was used to obtain extracts for metabolite profiling by gas chromatography-mass spectrometry (GC-MS) as described by Fiehn et al. (2000). Data analysis was performed using MassHunter Software, version B.07.00 (Agilent). For relative quantification, all metabolite peak areas were normalized to the peak area of the internal standard ribitol added prior to extraction and the amount of leaf material. From each sample, two extracts were prepared and analysed. \ No newline at end of file diff --git a/assays/CMML_18-0008_HPLC-DAD/isa.assay.xlsx b/assays/CMML_18-0008_HPLC-DAD/isa.assay.xlsx index 562882d253cf2d1158f8f286b55c7dd7628420fb..a1ffb28027dc7ec8edc23ba3de96b3fa472e4abc 100644 Binary files a/assays/CMML_18-0008_HPLC-DAD/isa.assay.xlsx and b/assays/CMML_18-0008_HPLC-DAD/isa.assay.xlsx differ diff --git a/assays/CMML_18-0008_LCqTOF-MasshunterQuant/isa.assay.xlsx b/assays/CMML_18-0008_LCqTOF-MasshunterQuant/isa.assay.xlsx index e93cd0a823866cc2abea343463f66b7fb9029ca0..570da59b644100c71f7e857d193c5c3887cde825 100644 Binary files a/assays/CMML_18-0008_LCqTOF-MasshunterQuant/isa.assay.xlsx and b/assays/CMML_18-0008_LCqTOF-MasshunterQuant/isa.assay.xlsx differ diff --git a/assays/CMML_18-0008_LCqTOF/isa.assay.xlsx b/assays/CMML_18-0008_LCqTOF/isa.assay.xlsx index 660e9b2d36de9657ea1c7c31d2445eb05d0a92a3..25bc67bb8441c57daf66b36941a18bdb18e89953 100644 Binary files a/assays/CMML_18-0008_LCqTOF/isa.assay.xlsx and b/assays/CMML_18-0008_LCqTOF/isa.assay.xlsx differ diff --git a/assays/Maleckova2018_aba_mRNASeq/isa.assay.xlsx b/assays/Maleckova2018_aba_mRNASeq/isa.assay.xlsx index 1f03dc0eb0e84c70177377738e63e95068440a7b..9f07a0952216bbfccb6ce07ff34eda0a76d5ad20 100644 Binary files a/assays/Maleckova2018_aba_mRNASeq/isa.assay.xlsx and b/assays/Maleckova2018_aba_mRNASeq/isa.assay.xlsx differ diff --git a/assays/ProteinAndStarchMeasurements/README.md b/assays/ProteinAndStarchMeasurements/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/ProteinAndStarchMeasurements/dataset/.gitkeep b/assays/ProteinAndStarchMeasurements/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/ProteinAndStarchMeasurements/isa.assay.xlsx b/assays/ProteinAndStarchMeasurements/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..c0caffd0cbbc2d8497868468a4b4d2b22cf79a7b Binary files /dev/null and b/assays/ProteinAndStarchMeasurements/isa.assay.xlsx differ diff --git a/assays/ProteinAndStarchMeasurements/protocols/.gitkeep b/assays/ProteinAndStarchMeasurements/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/ProteinAndStarchMeasurements/protocols/ProteinAndStarchMeasurementsProtocol.md b/assays/ProteinAndStarchMeasurements/protocols/ProteinAndStarchMeasurementsProtocol.md new file mode 100644 index 0000000000000000000000000000000000000000..5890e9eff0b1df0f67eb025a22930c0e538fe196 --- /dev/null +++ b/assays/ProteinAndStarchMeasurements/protocols/ProteinAndStarchMeasurementsProtocol.md @@ -0,0 +1,5 @@ +## Protein and Starch Measurements + +Approximately 25 mg of ground leaf material was used for extraction in 80% (v/v) ethanol with 10 mM 2-(N-morpholino)ethanesulfonic acid (pH 6). Extraction was performed three times with 500 µl extraction buffer while shaking for 1 hour at 90 °C. After each extraction step, samples were centrifuged at 12,000 g for 10 min and the supernatant was collected and pooled with previous fractions. From each sample, two extracts were prepared and stored at –20 °C until analysed. The protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). + +Pellets obtained during protein extraction were gelatinized with 300 µl 0.2 M NaOH at 90 °C for 40 minutes. After adjustment to pH 5–6, 200 µl hydrolysis buffer was added [20 mM acetate buffer, pH 4.8; 0.5 U α-amylase (10102814001, Roche), and 4 U amyloglucosidase (A7095, Roche)] and the mixture was incubated overnight at 37 °C. Glucose resulting from starch degradation was measured enzymatically: 10 µl of digest was added to 100 mM HEPES-NaOH (pH 7.5) with 10 mM MgCl2, 1 mM NADP+, 2 mM ATP, 0.1 U glucose-6-phosphate dehydrogenase (G6PDHII-RO, Roche), and 4 U hexokinase (11426362001, Roche). The final reaction volume was 200 µl and the increase of absorbance at 340 nm was monitored. \ No newline at end of file diff --git a/assays/TitratableAcidity/README.md b/assays/TitratableAcidity/README.md new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/TitratableAcidity/dataset/.gitkeep b/assays/TitratableAcidity/dataset/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/TitratableAcidity/isa.assay.xlsx b/assays/TitratableAcidity/isa.assay.xlsx new file mode 100644 index 0000000000000000000000000000000000000000..66d08a9bbbe61e83caf871596a587ec278c26184 Binary files /dev/null and b/assays/TitratableAcidity/isa.assay.xlsx differ diff --git a/assays/TitratableAcidity/protocols/.gitkeep b/assays/TitratableAcidity/protocols/.gitkeep new file mode 100644 index 0000000000000000000000000000000000000000..e69de29bb2d1d6434b8b29ae775ad8c2e48c5391 diff --git a/assays/TitratableAcidity/protocols/TitratableAcidityProtocol.md b/assays/TitratableAcidity/protocols/TitratableAcidityProtocol.md new file mode 100644 index 0000000000000000000000000000000000000000..15a5cbc8c8427cb11b54a5726a2c2a00a2d4eb9a --- /dev/null +++ b/assays/TitratableAcidity/protocols/TitratableAcidityProtocol.md @@ -0,0 +1,3 @@ +## Titratable acidity + +Approximately 30 mg of ground leaf material was incubated in 500 µl 50% (v/v) methanol at 90 °C while shaking. After centrifugation at 13,000 g for 5 min at room temperature, 450 µl of the supernatant was collected and the remaining pellet was extracted once more. Both fractions were pooled and stored at –20 °C until use. Titratable acidity was measured using bromothymol blue (Carl Roth) as a pH indicator: 60 µl of extract was mixed with 40 µl distilled H2O and 4 µl bromothymol blue (1 mg/ml stock). Using the microinjector of a Synergy H1 (BioTek) plate reader, samples were titrated with 1 mM KOH in 5 µl steps. After each step, absorbance at 445 nm and 615 nm was recorded, and the ratio of the absorbances at 615/445 nm was calculated. Based on the 615/445 ratio of buffer standards (pH 4.6–7.5), the pH of analysed extracts was determined. From the volume of KOH required to titrate extracts to pH 7.0, the titratable acidity was calculated. Two extractions were performed from each sample, and each extract was titrated in three replicates. \ No newline at end of file diff --git a/studies/Maleckova2018_aba/isa.study.xlsx b/studies/Maleckova2018_aba/isa.study.xlsx index 71b351376e487b5e5c895ca49d335025e10e957f..1e2002e66314c0909db007dd8328c963670ee237 100644 Binary files a/studies/Maleckova2018_aba/isa.study.xlsx and b/studies/Maleckova2018_aba/isa.study.xlsx differ