Bursaphelenchus xylophilus  YW4  strain at phytophagous   stage (12 h postinfection)
1)mRNA enrichment,the mRNA with Polya tail was enriched by magnetic beads with oligodt; rRNA removal: the rRNA was hybridized with DNA probe, the DNA / RNA hybrid strand was selectively digested by RNaseH, and then the DNA probe was digested by DNaseI to obtain the required RNA after purification.2) The obtained RNA was segmented by using the interrupt buffer, then the random N6 primers were used for reverse transcription, and then the cDNA double strand was synthesized to form double stranded DNA.3) The end of the synthesized double stranded DNA was flattened and phosphorylated at the 5 'end. The 3' end formed a sticky end protruding "a", and then connected with a bubbly junction protruding "t" at the 3 'end.4) The ligation products were amplified by PCR with specific primers.5) PCR products were heat denatured into single strand, and then cyclized with a bridge primer to obtain single strand circular DNA library.6) Sequencing.
SUB9273662
Bx-Z