RNA-Seq of developing pericarp and embryo of purple and brown pericarp rice
Total RNA was isolated from 10 day developing embryo and pericarp samples.  RNA concentration and integrity was verified using BioAnalyzer 2100 with RNA 6000 Nano Labchips according to manufacturers instructions (Agilent Technologies). RNA samples had 28S/18S ratios ranging from 1.8 to 2.0 and RIN (RNA Integrity Number) values of between 7.0 and 9.0. PolyA RNA was isolated from 510 ug total RNA with oligo(dT) beads using two rounds of oligo-dT purification and 50100 ng mRNA was used for Illumina library preparation. The directional cDNA libraries were prepared using the dUTP method with a NEB Ultra directional RNA Library Prep kit for Illumina (New England Biolabs Inc). Each sample was ligated with different indexes and amplified with 12 PCR cycles. The quality and quantity of cDNA libraries were evaluated using Qubit 2.0 (Invitrogen, Life Technologies), BioAnalyzer 2100 with DNA 1000 kit (Agilent Technologies) and Real-time PCR using KAPA Library Quantification Kit (Kapa Biosystems). Adapter-ligated cDNA fragment libraries were pooled together and loaded into an Illumina NextSeq500 using NextSeq 500/550 High Output Kit version 3 according to the manufacturers protocol (Illumina).