RNA-Seq of mus musculus: adult male hepatic macrophage
The mRNA with polyA tail was enriched by Oligo(dT) magnetic beads, and then randomly interrupted by divalent cations in the NEB Fragmentation Buffer. Using fragmented mRNA as template and random oligonucleotides as primers, the first strand of cDNA was synthesized in M-MuLV reverse transcriptase system, and then the RNA strand was degraded by RNaseH. The second strand of cDNA was synthesized by dNTPs in the system of DNA polymerase I. The purified double-stranded cDNA underwent terminal repair. After terminal repair, A-tail was added and sequencing joints were connected. cDNA of about 200bp was screened by AMPure XP beads. PCR amplification was performed, and PCR products were purified by AMPure XP beads again, and finally a library was obtained.