Name: MN18091_p
Instrument: NextSeq 550
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: PolyA
Layout: PAIRED
Construction protocol: Samples from 10 tissues throughout development were collected to generate expression evidence for gene annotation. Two biological replicates were collected for each tissue type, and each replicate consisted of three individual plants. The tissues that were sampled were: 1) primary root at six days after planting; 2) shoot and coleoptile at six days after planting; 3) base of the 10th leaf at the Vegetative 11 (V11) growth stage; 4) middle of the 10th leaf at the V11 growth stage; 5) tip of the 10th leaf at the V11 growth stage; 6) meiotic tassel at the Vegetative 18 (V18) growth stage; 7) immature ear at the V18 growth stage; 8) anthers at the Reproductive 1 (R1) growth stage; 9) endosperm at 16 days after pollination; and 10) embryo at 16 days after pollination. Tissue from developmental stage V11 and older were taken from field-grown plants while all younger tissue samples were taken from greenhouse-grown plants. For the endosperm and embryo samples, tissue from 50 kernels per plant (150 total per biological replicate) were sampled. Greenhouse-grown plants were planted in Metro-Mix300 (Sun Gro Horticulture) with no additional fertilizer and grown under greenhouse conditions (27°C/24°C day/night and 16h/8h light/dark) at the University of Minnesota Plant Growth Facilities. Field grown plants were planted at the Minnesota Agricultural Experiment Station located in Saint Paul, MN with 30-inch row spacing at ~52,000 plants per hectare. RNA was extracted using the Qiagen RNeasy plant mini kit following the manufacturer's suggested protocol. The quality of the total RNA was assessed by Bioanalyzer or Fragment analyzer to determine RNA concentration and integrity. The sample concentration was normalized in 25 uL of nuclease-free H2O before library preparation. Libraries were prepared using KAPA's stranded mRNA-seq kit with halved reaction volumes. During library preparations, mRNA was selected using oligo-dT beads, the RNA was fragmented, and cDNA was generated using random hexamer priming. Single or dual indices were ligated depending on the desired level of multiplexing. The number of cycles for library PCR was determined based on kit recommendations for the amount of total RNA used during library preparation. Libraries were quality control checked using Qubit or plate reader, depending on the number of samples in the batch for library concentration, and fragment analyzer for the size distribution of the library. The pooling of samples was based on qPCR. The pooled libraries were then checked by Qubit, Fragment Analyzer, and qPCR.