RNA-Seq of human blood: adult normal female and male
After the RNA sample is qualified, remove the rRNA from the total RNA sample (part of the lncRNA has the same polyA tail structure as the mRNA, and the lncRNA containing polyA tail can be retained to the greatest extent by removing the rRNA). Subsequently, fragmentation buffer was added to the enriched RNA to break the RNA into small fragments. Using the fragmented RNA as a template, 6nt random primers were added to reverse transcribe and synthesize the first strand of cDNA, and then buffer, dNTPs(dNTP in DNTP was replaced by dUTP), DNA polymerase I and RNase H were added to synthesize the second strand of cDNA. After purification, terminal repair, adding A and connecting sequencing linker, the synthesized double-stranded cDNA was degraded by USER enzyme and enriched by PCR. Finally, the PCR product was purified by AMPure XP beads to obtain the final chain-specific library.