RNA-seq of Phomopsis liquidambaris B3: pure culture
Extract total RNA, use Oligo(dT) magnetic beads adsorption to purify mRNA, and then use Oligo(dT) as primer to synthesize cDNA. The 5' ends of tags can be generated by two types of Endonuclease: Nla or Dpn (see table 1 for recognition sites). Usually, the bead-bound cDNA is subsequently digested with restriction enzyme Nla, which recognizes and cuts off the CATG sites. The fragments apart from the 3' cDNA fragments connected to Oligo(dT) beads are washed away and the Illumina adaptor 1 is ligated to the sticky 5' end of the digested bead-bound cDNA fragments. The junction of Illumina adaptor 1 and CATG site is the recognition site of Mme, which is a type of Endonuclease with separated recognition sites and digestion sites. It cuts at 17bp downstream of the CATG site, producing tags with adaptor 1. After removing 3' fragments with magnetic beads precipitation, Illumina adaptor 2 is ligated to the 3' ends of tags, acquiring tags with different adaptors of both ends to form a tag library. After linear PCR amplification, fragments are purified by PAGE Gel electrophoresis. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library. At last, the library could be sequenced using Illumina HiSeq 2000 or other sequencer when necessary.