GSM4040730: H25D1; Zea mays; RNA-Seq
developing seeds were collected manually at 15-, 20-, 25-, 30-, 35- and 40-DAP in triplicate. Seeds were soaked in water overnight at room temperature and embryos were then dissected from their endosperm and frozen immediately using liquid nitrogen and stored at -80 °C until use. Thirty six libraries representing the embryos from the two genotypes collected at six developmental stages and in three replicates were constructed for transcriptome sequencing. Total RNA was extracted using a TIANGEN RNA Prep Pure Plant kit (Tiangen Biotech Co. Ltd, Beijing, China), and purified with the Dynabeads® Oligo (dT)25 kit (Life, USA). RNA concentration was measured using nanodrop spectrophotometer (Nanodrop Technologies, USA) and integrity was evaluated with the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The cDNA library was constructed by using a NEBNext® Ultra RNA Library Prep Kit, the quality of retrieved cDNA was checked using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA). After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then, 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. Sequencing was perfomed using an Illumina HiSeq™ 2500 paired-end sequencing system.