rice RNAseq project
The rice RNA samples were sent to BGI (The Beijing Genomics Institute at Shenzhen, China) for sequencing. The mRNA of every sample was enriched by using oligo(dT) magnetic beads. The mRNA was interrupted to short fragments (about 200 bp). First strand cDNA was synthesized by random hexamer-primers using the mRNA fragments as templates. Buffer, dNTPs, RNase H and DNA polymerase I were added to synthesize the second strand. The double strand cDNA was purified with a QiaQuick PCR extraction kit and washed with EB buffer for end repair and single nucleotide A (adenine) addition. Finally, sequencing adaptors were connected to the fragments. The fragments were purified by agrose gel electrophoresis and enriched by PCR amplification. The library products were then ready for sequencing analysis via Illumina HiSeq™ 2000.     When rice seedlings grew to three-leaf stage old,1g of leaves were collected equally from the selected four (dx, cl, 93-11) or 20 (OOP) plantlets from each of the four experimental materials for RNA isolation, both prior to and following the cold treatment.