title: miRNA-Seq of larvae Toxocara canis in RPMI-1640 with plasma 3
design: Process the total RNA by mRNA enrichment or rRNA removal. mRNA enrichment: use magnetic beads with OligodT to enrich mRNA with polyA tails; rRNA removal: hybridize the rRNA with a DNA probe, selectively digest the DNA/RNA hybridization strand with RNaseH, and then use DNaseI to digest the DNA probe, and then purify it to obtain the desired RNA. After purification, the desired RNA is obtained. Fragment the RNA with interrupted buffer, reverse transcribe with random N6 primer, and synthesize the cDNA duplex to form double-stranded DNA.The synthesized double-stranded DNA is flattened and phosphorylated at the 5' end, a sticky end with a protruding "A" is formed at the 3' end, and a bulge-like junction with a protruding "T" at the 3' end is attached. The ligated product is amplified by PCR with specific primers. The PCR product is heat denatured into a single strand, and then the single-stranded DNA is cyclized with a bridge primer to obtain a single-stranded circular DNA library. Sequencing.