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To detect changes in the global leaf and MSA transcriptomes in response to HT, we harvested at ZT14 the leaves and developing MSAs of GP and GP-fast grown under LD either under CT or HT at four developmental stages, W1.0, W2.0, W3.5, and W6.0 for RNA-sequencing. Each replicate of leaf samples was collected by pooling the latest fully elongated leaf on the main culm from three plants. Each replicate of MSA samples was collected by pooling ca. 30, 20, 15, and 10 MSAs from plants at stages W1.0, W2.0, W3.5, and W6.0, respectively. All MSA samples were collected under a stereo microscope in the environment where the plant grew. A total of three to four biological replicates of MSA and leaf samples were used for RNA-sequencing.
Total RNA was extracted from MSA and leaf samples using the RNeasy Plant Mini Kit (Qiagen, Germany) with beta-mercaptoethanol added, following the manufacturer’s instructions, and followed by the digestion with DNase I (Qiagen, Germany). RNA samples passing a cutoff of RNA Integrity Number (RIN) ≥ 6 were used for mRNA library preparation with poly(A)-enrichment. Paired-end 150 bp sequencing was performed on NovaSeq 6000 sequencing platform, and at least 5G of clean reads data per sample were generated by Novogene Co., Ltd (UK). All samples passed the assessment of the adaptor and GC contents by FastQC (Andrews 2010).
