GSM4007576: globular stage embryo proper; Arabidopsis thaliana; RNA-Seq
Total RNA was extracted using the RNaqueous Micro-Kit (Ambion, Austin, TX) using a modified protocol incorporating an on-column DNase treatment (Qiagen RNase-Free DNase Set) during column washes. Amplification was performed using NuGen Ovation Pico WTA V1 (San Carlos, CA), starting with 15ng total RNA. Double-stranded cDNA was generated using the NuGen WT-Ovation Exon-Module (San Carlos, CA). Illumina-complatible libraries were made starting with 150 ng double-stranded cDNA using Kumar et al. (Kumar R, Ichihashi Y, Kimura S, Chitwood DH, Headland LR, Peng J, Maloof JN and Sinha NR (2012) A high-throughput method for Illumina RNA-Seq library preparation. Front. Plant Sci. 3:202. doi: 10.3389/fpls.2012.00202) Supplemetary Methods 1 library preparation protocol, starting at Day 2. The following modifications were done: the Illumina TruSeq DNA Sample preparation kit adapters were used (1/25 dilution) and the Agilent Pfu Turbo Cx hotstart DNA polymerase was used for the PCR enrichment step (15 cycles). Libraries were quantified using Quant-iT PicoGreen dsDNA Reagent (Grand Island, NY) and a Nanodrop ND-3300 instrument.