Dietzia sp.111N12-1 transcriptomic
The cDNA library for Illumina sequencing was prepared following the instruction of the TruseqTM RNA sample prep Kit (Illumina). First, mRNA was isolated from the total quantified RNA using poly-T oligo-attached magnetic beads (Illumina). The mRNA was subsequently disrupted into fragments (200-700 nt) using divalent cations. First-strand cDNAs were then synthesized using SuperScript II reverse transcriptase with small-fragment RNAs as templates and second-strand cDNA synthesis by DNA polymerase I. Next, these cDNA fragments were end-repaired and 3’-adenylated using T4 DNA polymerase, Klenow DNA polymerase, and T4 polynucleotide kinase. Illumina’s paired-end adaptors were ligated to the ends of these 3’-adenylated cDNA fragments. The cDNA fragments with adaptors were enriched by 15 cycles of PCR amplification with Phusion DNA polymerase, and the products were electrophoresed on a 2%-agarose gel (certified low-range, ultra agarose), with 200-bp (± 25 bp) fragments purified for downstream sequencing.