Illumina HiSeq 2000 sequencing; GSM1384330: Globular stage embryo proper-biological replicate 1; Phaseolus vulgaris; RNA-Seq

Globular stage embryo proper-biological replicate 1

We used laser capture microdissection (LCM) to capture globular stage common bean embryo proper and suspensor, and profiled the transcriptome of these two embryo regions using next-generation sequencing. Our long-term goal is to understand the region-specific differentiation processes that occur during early embryo development and how genes are activated specifically in the suspensor and embryo proper. Overall design: Illumina sequencing of transcripts from laser-captured embryo proper and suspensor regions of common bean globular stage embryo. Two biological replicates were collected for each embryo region.

Transcript Profiling of Common Bean Embryo Regions at Globular Stage Using Laser Capture Microdissection Coupled with RNA-Seq

Submitted by Gene Expression Omnibus on 23-MAY-2014

Six days after pollination, seeds with length between 1.6mm and 2.0mm  and containing globular-stage embryos were fixed in 3:1(v/v) ethanol:acetic acid and embedded in paraffin (Kerk et al., Plant Physiol.132. 27-35 (2003)). Embryo regions were captured from six-micron sections using a Leica DM6000 system. Total RNA was extracted using the PicoPure RNA Extraction Kit (Molecular Devices, Sunnyvale, CA). Five nanograms of total RNA was converted to complementary DNA (cDNAs) and amplified using the Nugen Ovation RNA-Seq kit. Approximately 500 nanograms of cDNA was used as a template for blunt end repairs, 3' A Tailing and adapter ligation following the Illumina mRNA-Seq protocol. Purified cDNAs were amplified by PCR for 10 cycles. PCR-amplified cDNA fragments were size selected using the AMpure XP beads (Beckman). Quantification of cDNA libraries was carried out using a Nanodrop ND-3300 Fluoro-spectrophotometer (Thermo Scientific, Waltham, MA). Phi-X174 DNA was spiked in to the library by the sequencing facility before cluster formation and sequencing.