title: GSM4669828: E_B60_33R3; Zea mays; RNA-Seq
library_construction_protocol: RNA was extracted from small samples (0.1 g) of leaf lamina (avoiding the midrib) obtained from the middle of the first fully expanded leaves. The samples were taken in triplicate, one sample from each of three different plants, in the two chambers set for the same conditions.  The samples obtained from the plants in the two different chambers were taken at the same time of virtual day, because the temperature and light cycles are offset to allow for sampling at the same time of day. The samples were separately ground to a powder in liquid nitrogen, and RNA was isolated using a Plant RNeasy Mini Kit and treated with DNase (Qiagen, www.qiagen.com/us/) according to the manufacturer's instructions. RNA quality and quantity were determined by the UV absorbance spectra, gel electrophoresis and Agilent 2000 BioAnalyzer (http://www.agilent.com). For RNAseq analysis, poly A RNA was isolated from the purified RNA using oligo(dT)-attached magnetic beads and fragmented at elevated temperature with NEBNext RNA fragmentation buffer (New England BioLabs).  cDNA library construction, sequencing, and primary bioinformatics analysis were performed by BGI Tech Solutions Co., Ltd. (Beijing Genomic Institute (www.genomics.org.cn, BGI)) as follows:  First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by second-strand cDNA synthesis.  cDNA ends were repaired by A-Tailing followed by adding RNA Index Adapters. The cDNA fragments were amplified by PCR, and the products purified by Ampure XP Beads (Beckman Coulter).  The products were validated for quality control using an Agilent Technologies 2100 bioanalyzer. The double stranded PCR products were heat denatured and circularized using splint oligos (Diegelman and Kool, 2000). The single strand circular DNAs (ssCir DNAs) were amplified with phi29 (Blanco et al., 1989) to make DNA nanoballs (DNBs) (Porreca, 2010).  DNBs were loaded into the patterned nanoarray and single end 50 base reads were generated on BGIseq500 platform (BGI-Shenzhen, China).
attributes.tag: GEO Accession
attributes.value: GSM4669828