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RNA of milk cells were extracted from the milk of dairy cows,Ribosomal RNA is removed from the total RNA, resulting in mRNA. Subsequently, the mRNA fragments were randomly interrupted with divalent cations in NEB Fragmentation Buffer. The first cDNA strand was synthesized in M-MuLV reverse transcriptase system with the fragment mRNA as template and random oligonucleotides as primer. Then the RNA strand was degraded by RNaseH, and the second cDNA strand was synthesized by dNTPs in the DNA polymerase I system. The purified double-stranded cDNA was end-repaired, A-tail was added, and sequencing joints were connected. cDNA of about 250-300bp was screened by AMPure XP beads for PCR amplification, and PCR products were purified by AMPure XP beads again, and finally A library was obtained. |
RNA-Seq |
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PCR |
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