NextSeq 500 paired end sequencing; GA20-oxidase overexpression and heterosis stimulate complementary growth mechanisms
GA20-oxidase overexpression and heterosis stimulate complementary growth mechanisms
Mo17-pool3_p
Samples were taken from 3 parental lines and 2 hybrid crosses. The parents: inbred lines B104 and Mo17, and the GA20OX transgenic line in B104 background. The hybrid crosses: B104 x Mo17, GA20OX x Mo17. The first 0.5 cm (dividing cells) of the most basal part of leaf four of at 2 days after leaf appearance was sampled from the parental lines and hybrid crosses. For each genotype, there are 3 biological replicates, each pool consisting of proliferative tissue of three plants. After harvesting, samples were directly frozen in liquid nitrogen. To sample the most basal half centimeter of the fourth leaf, the older leaves that surround the growing leaf should be removed. Special care should be taken not to damage the basal part of the growing leaf because this is where the leaf basal meristem is located.  Growth chambers have a controlled relative humidity (55%), temperature (24C), and light intensity  (170 mmol m-2 s-1 photosynthetically active radiation at plant level) in a 16-h/8-h day/night rhythm  provided by a combination of high-pressure sodium vapour (RNP-T/LR/400W/S/230/E40; Radium)  and metal halide lamps with quartz burners (HRI-BT/400W/D230/E40; Radium).  Total RNA was isolated from basal half cm of growing leaf 4 with the guanidinium thiocyanate-phenol-chloroform extraction method using TRI-reagent (Sigma-Aldrich). RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000  (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent).  Per sample, 500 ng of total RNA was used as input. Using the Illumina TruSeq Stranded mRNA  Sample Prep Kit (protocol 15031047 Rev E October 2013) poly-A containing mRNA molecules were  purified  from  the  total  RNA  input  using  poly-T  oligo-attached  magnetic  beads.  In  a  reverse  transcription  reaction  using  random  primers,  RNA  was  converted  into  first  strand  cDNA  and  subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction. The  cDNA fragments were extended with a single A base to the 3 ends of the blunt-ended cDNA  fragments after which multiple indexing adapters were ligated introducing different barcodes for  each sample. Finally, enrichment PCR was carried out to enrich those DNA fragments that have  adapter molecules on both ends and to amplify the amount of DNA in the library. For the sequence  run, libraries were equimolarly pooled and sequenced using a high 300 cycles (PE- 2 x 150 bp)  NextSeq kit on an Illumina NextSeq 500 instrument.