Illumina HiSeq 2500 paired end sequencing; RNAseq analysis on mature leaf tissue of 60 individual B104 maize inbred plants grown in the field
RNAseq analysis on mature leaf tissue of 60 individual B104 maize inbred plants grown in the field
In total, 202 representative plants at VT stage that exhibited a primary ear at leaf 16 were selected. Damaged plants and border plants were excluded to avoid large perturbations and discrimination of groups based on the level of perturbation during analysis. Subsequently, the primary ear leaf was cut off from the plant at the ligule and 10cm mature leaf tissue in the middle of leaf 16 blade of the 202 selected plants was sampled in liquid nitrogen on the field. During sampling the midrib of the blade was removed to avoid detection of exogenous metabolites during untargeted metabolite profiling. Sampling occurred from 10 am in the morning till noon. Subsequently, all samples were grinded and randomly select 60 individual leaf samples for transcriptome analysis. To sample 10cm mature leaf tissue in the middle of leaf 16 blade scissors were used. During the summer of 2015, B104 maize inbred plants were grown under 'uncontrolled' field conditions in Belgium at our site in Zwijnaarde (51°00'35.2\"N, 3°42'56.5\"E) with a sowing density of approximately 177.778 plants per hectare. The planting scheme consisted of one block with ten adjacent rows of B104 plants of 6 m each (containing 56 plants) and spaced 75 cm apart. North and West of the B104 plants a commercial hybrid, Ricardinio, was sown as border plant surrounding the field trial while in East and South directions hybrids and recombinant inbred lines were neighboring. No treatments were applied. Total RNA was extracted using the guanidinium thiocyanate-phenol-chloroform extraction method using TRI-reagent (Thermo Fisher Scientific) followed by DNA digestion using the RQ1 RNase-free DNase kit (Promega). Total RNA was sent to GATC Biotech for RNA sequencing. Library preparation was done using the NEBNext Kit (Illumina). In brief, purified poly(A)-containing mRNA molecules were fragmented, randomly primed strand-specific cDNA was generated and adapters were ligated. After quality control using an Advanced Analytical Technologies Fragment Analyzer, clusters were generated through amplification using cBOT (Cluster Kit v4, Illumina)
Experimental Factor: individual
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