The RNA-seq transcriptome of Aspergillus flavus CA43 mycelia
Total RNA of each sample was extracted using RNAisoTM Plus (TaKaRa), treated with RNase-free DNase I (TaKaRa) and purified by NucleoSpin® RNA Clean-up Kit (Macherey-Nagel). RNA integrity was assayed using Agilent Technologies 2100 Bioanalyzer. Sera-mag magnetic Oligo(dT) beads were used to isolate poly(A)-mRNA from 20 µg purified total RNA. mRNA were eluted by 10 mmol/L Tris buffer and fragmented into small pieces in the range of 100 bp-500 bp by divalent cations treatment. Taking these short fragments as templates, random hexamer-primers were used to synthesize the first-strand cDNA using SuperScript II, RNaseH and DNA polymerase I. First-strand cDNA was purified using QIAquick PCR Purification Kit and used as the template for synthesizing dUTP-containing second-strand cDNA by adding buffer, dATP, dCTP, dGTP, dUTP, RNAseH and DNA polymerase I (dUTP was used here instead of dTTP). Double-strand cDNA fragments were purified with QIAquick PCR Purification Kit and dissolved with elution buffer for end-repair, phosphorylation and 3’-adenylation. After that, cDNA fragments were purified with Qiagen MinElute PCR Purification Kit. Illumina sequencing adapters were ligated to the 3’-adenylated cDNA fragments, followed by purification using MinElute PCR Purification Kit. Certain fragments with length of 100 bp -500 bp were purified using QIAquick Gel Extraction Kit from the above modified double-strand cDNA fragments separated by 2% TAE-agarose gel (Certified Low-Range Ultra Agarose, Biorad). Uracil N-Glycosylase (UNG) was added to digest dUTP-containing second-strand cDNA at alkalescent medium and high temperature. The remaining first-strand cDNA was purified using MinElute PCR Purification Kit, and enriched by fifteen rounds of PCR amplification using Primer P7 and Primer P5 (which were homologous to the Illumina sequencing adapters). After purification using QIAquick Gel Extraction Kit, fragments of 100 bp-500 bp (average 200 bp) were selected as strand-specific RNA-Seq cDNA library. Finally, after quantification on an Agilent Technologies 2100 Bioanalyzer, the cDNA library were sequenced using Primer P7 and P5 on Illumina HiSeq™ 2000 platform.
SCUT
Aflavus_CA43_M