Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: qiagen Rneasy RNA extraction Beads with oligo(dT) were used to isolate poly(A) mRNA after total RNA was collected from eukaryote (prokaryocyte can be treated with kit to remove rRNA before next step). Fragmentation buffer was added for interrupting mRNA to short fragments. Taking these short fragments as templates, Random hexamer-primer were used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNase H and DNA polymerase I, respectively. Short fragments were purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly(A). After that, the short fragments were connected with sequencing adaptors. And, for amplification with PCR, we selected suitable fragments, as templates, with respect to the result of agarose gel electrophoresis. At last, the library could be sequencing using Illumina HiSeq™ 2000.