RNA-Seq of Homo sapiens: Diffuse large B cell lymphoma
Treatment of total RNA by mRNA enrichment or rRNA removal, mRNA enrichment: enrichment of mRNA with polyA tail using magnetic beads with OligodT; rRNA removal: hybridization of rRNA with DNA probe, selective digestion of DNA with RNaseH /RNA hybridization chain, and then DNA probe is digested with DNaseI, and purified to obtain the desired RNA. The obtained RNA is fragmented by interrupting the buffer, and the random N6 primer is used for reverse transcription, and then the cDNA double strand is synthesized to form double-stranded DNA. The synthetic double-stranded DNA ends are flattened and phosphorylated at the 5' end, and the 3' end forms a sticky end protruding "A", and then a bubbling joint having a convex "T" at the 3' end is attached. The ligation product is PCR amplified by specific primers. The PCR product is heat-denatured into a single strand, and a single-stranded DNA is cyclized with a bridge primer to obtain a single-stranded circular DNA library.