miRNA-Seq of Pinctada fucata martensii:mantle central
1) Filter small RNA: separate 18-30nt RNA segment by PAGE gel.  2) 3' adaptor ligation: link 5-adenylated, 3-blocked single-stranded DNA adapter to 3' end of selected small RNAs from step 1.  3) Reverse primer annealing: the RT primer is added to the solution from step 2, and is cross-linked to the 3' adapter of RNAs and excessive free 3' adapter.  4) 5' adaptor ligation: the 5' adaptor is linked to the 5' end of the product from step 3. The connection (5 adaptor) will be attached to the 5 end only, but it is not c onnected to the 3' adaptor or RT primer hybrid chain. This greatly reduces self-ligation.  5) Strand cDNA synthesis: reverse extend the RT primer in step 3 to synthetize strand cDNA.  6) PCR amplification: use high-ping polymerase to amplify cDNA, enrich cDNA with both 3' and 5' adaptor.  7) Library fragment selection: separate the PCR product of 100~120bp by PAGE gel to eliminate primer-dimer and other byproducts.  8) Library quantitative and pooling cyclization.