assays/ThreeDRNAseq/protocols/NormalizationWithThreeDRNAseq.md

+## Normalization with ThreeDRNAseq R package
+
+We kept transcripts with a minimum of 1 CPM (counts per million) in at least three samples. Normalization of TPM (transcript per million) was conducted using the ‘ThreeDRNAseq’ R package (v2.0.1) (Guo et al., 2021). For TPM values, see Supplementary Dataset 2 and 3. As the samples were collected at different DAEs and RNA extraction was conducted in several batches, we removed the batch effect in the gene count by using the ThreeDRNAseq’ R package (v2.0.1) (Guo et al., 2021).
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studies/plant-samples/protocols/PlantMaterialsAndGrowthConditions.md

+## Plant Materials and Growth Conditions
+
+In this study, we selected two genotypes carrying a wild-type *Ppd-H1* allele and three genotypes with mutant *ppd-h1* allele to study the interaction between *Ppd-H1* and HT on shoot growth and inflorescence development. Golden Promise (GP) and Scarlett are spring barley cultivars carrying a natural mutation in the CCT domain of *PPD-H1* (G>T at CDS position 1969), according to gene model HORVU.MOREX.r3.2HG0107710.1 (Supplementary Dataset 1), causing a Gly-to-Trp amino acid exchange, and resulting in a late flowering time under long-day (LD; 16 h/8 h, day/night) conditions. GP-fast and S42-IL107 are two near-isogenic lines (NILs) derived from GP and Scarlett, respectively, carrying wild-type Ppd-H1 alleles from winter barley Igri and wild barley (*Hordeum vulgare ssp. spontaneum*) accession ISR42-8 (Supplementary Dataset 1). These lines are early flowering under LD (Schmalenbach et al., 2011; Digel, Pankin and von Korff, 2015; Gol, Haraldsson and Von Korff, 2021).
+
+Plants were sown in a 96-well growing tray with ED73 soil (Einheitserde Werkverband e.V., Germany) mixed with 7% sand and 4 g/L Osmocote Exact Hi.End 3-4M (ICL Group Ltd.). Seeds were stratified for three days at 4 °C in the soil for even germination and then transferred to controlled growth chambers (PAR 300 µM/m2s, humidity 60%) with inductive LD conditions (16 h/8 h, day/night) set to control (CT; 20 °C/16 °C, day/night) or high ambient temperature (HT; 28 °C/24 °C, day/night).
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