title: Comparative analysis reveals dynamic changes in miRNAs and their targets and expression during somatic embryogenesis in longan (Dimocarpus longan Lour.).
design: To identify potential conserved and novel miRNAs from longan somatic embryo, a pooled sRNA library was generated from the synchronized embryogenic cultures (friable-embryogenic calli, globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledonary embryos) and sequenced on a Solexa system (Illumina). First,small (16–30 nt) RNA fragments were isolated from a 15% PAGE gel and purified. The small RNAs were then sequentially ligated to a59 RNA adapter (59-GUUCAGAGUUCUACAGUCCGACGAUC-39) and a 39 RNA adapter (59-pUCG UAUGCCGUCUUCUGCUUGidT-39; p, phosphate; idT, inverted deoxythymidine) using T4 RNA ligase. The resulting samples were reversetranscribed to cDNA with an RT primer (59-CAAGCAGAAGACGGCATACGA-39) using Superscript II reverse transcriptase (Invitrogen) and PCR-amplified. Finally, Solexa sequencing was used to sequence the sRNAs in the pooled longan samples (Beijing Genomics Institute, China).