B16F10(Melanoma) cell line was treated-aMSH_2
After extracting the total RNA from samples, mRNA and non-coding RNAs are enriched by removing rRNA form the total RNA with kit, and you also can choose to remove mRNA by removing polyA RNAs. By using the fragmentation buffer, the mRNA and non-coding RNAs are fragmented into short fragments (about 200~700 bp), then the first-strand cDNA is synthesized by random hexamer-primer using the fragments as templates. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand cDNA. The double strand cDNA is purified with QiaQuick PCR extraction kit and then used for end-polishing. Sequencing adapters are ligated to the fragments , then the second strand is degraded using UNG(Uracil-N-Glyosylase)finally. The fragments are purified by Agarose gel electrophoresis and enriched by PCR amplification.