RNA-seq of Mus musculu: Isolation of primary hepatic stellate cells from normal female livers over a period of 6 weeks
The method for synthesizing the first strand of cDNA by reverse transcription is the same as that of the NEB standard library construction method. The difference lies in that when synthesizing the second strand, dTTP in the dNTPs is replaced by dUTP. Then, the cDNA ends are repaired, an A tail is added, sequencing adapters are ligated, and length screening is performed. Subsequently, the cDNA second strand containing U is first degraded using the USER enzyme, and then PCR amplification is carried out and the library is obtained.