Illumina HiSeq 1000 paired end sequencing; Genetic response of maize silk to pollen tubes growth in comparison to fungal invasion.
Genetic response of maize silk to pollen tubes growth in comparison to fungal invasion.
Plants were grown in a greenhouse under controlled conditions with 16 hours light of 26-28°C and 8 hours night of 18-20°C. Whole seeds of 11 days pollinated cobs were harvested, isolated and stored at -80°C. Total RNA was prepared by grinding tissues in liquid nitrogen and total RNA was extracted by TRIzol reagent (TRIzol Plus RNA Purification Kit, Ambion) and processed following the protocol provided by manufacturer. To remove DNA within extraction and purification procedures, total RNA was treated with DNase (Qiagen), then total RNA was quantified and visualized on a Bioanalyzer (Agilent). Library preparation and RNAseq were carried out as described in the Illumina TruSeq RNA Sample Preparation v2 Guide, the Illumina HiSeq 1000 System User Guide (Illumina, Inc., San Diego, CA, USA), and the KAPA Library Quantification Kit - Illumina/ABI Prism User Guide (Kapa Biosystems, Inc., Woburn, MA, USA). In brief, 500 ng of total RNA was used for purifying the poly-A containing mRNA molecules using poly-dT oligo-attached magnetic beads. Following purification, the mRNA was fragmented to an average insert size of 200-400 bases using divalent cations under elevated temperature (94°C for 4 minutes). The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. The resulting cDNA fragments subsequently went through an end repair process, the addition of a single 'A' base, the ligation of the adapters, and a purification step. Finally the cDNA libraries were created by PCR enrichment. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit. Equimolar amounts of each library were used for cluster generation on the cBot (TruSeq PE Cluster Kit v3).
Experimental Factor: seed structure
organism part
Experimental Factor: none
stimulus