The regulation of transcriptome responses in zebrafish embryo exposure to triadimefon
cDNA/Total RNA was extracted from 50 homogenized zebrafish embryos treated with and without TDF, respectively, by using Trizol regent (Invitrogen, USA) according to the manufacturer’s instruction and treated with RNase-free DNase I (Takara Biotechnology, China). RNA integrity was confirmed with a minimum RNA integrated number value of 8 by the 2100 bio analyzer (Agilent). Poly(A) mRNA was enriched with oligo-dT beads and then was fragmented. The cleaved RNA fragments were transcribed into first-strand cDNA using reverse transcriptase and random hexamer primers, which was followed by second- strand cDNA synthesis. The double stranded cDNA was further subjected to end-repair, phosphorylation, 3’-adenylation and adaptor ligation in sequence. Adaptor ligated fragments were selected according to the size (250 to 350 bp), and the desired range of cDNA fragments were excised from the gel. The fragments were enriched by PCR amplification. The quality and quantity of the two libraries (one library was treated with TDF but the other one did not) were determined g on an Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System. Subsequently, the cDNA library was sequenced on a flow cell using Illumina HiSeq2000.