The invention discloses a method for constructing a strand-specific
transcriptome library. The method comprises steps as follows: step 1), mRNA (massager ribonucleic acid) fragmentation
processing is performed; step 2), inverse transcription is performed: the fragmented mRNA 3' terminal is connected with an R3 joint with the known sequence; an RT primer and the R3 joint are subjected to annealing
pairing for inverse transcription, and a first strand of cDNA (complementary
desoxyribonucleic acid) is synthesized; step 3), cDNA 3' terminal marking is performed: after
RNA is removed, TdT (
terminal deoxynucleotidyl transferase) is adopted to add multiple dC basic groups to the cDNA 3' terminal, ddCTP (2',3'-dideoxycytidine-5'-triphosphate) is adopted for end closing,
pairing is performed with an F5G primer, and the first strand of cDNA is duplicated under the action of
polymerase; step 4), PCR (
polymerase chain reaction) amplification is performed; step 5), quality testing is performed. The fragmented mRNA is connected with the joint and then is synthesized, so that the synthetic efficiency and the homogeneity of cDNA are improved; after the first strand of cDNA is synthesized, multiple dC basic groups are added to the cDNA 3' terminal by adopting TdT and ddCTP is adopted for closing,
pairing is performed with the F5G primer containing multiple dG basic groups, the first strand of cDNA is duplicated under the action of
polymerase, therefore, production of self-linked dimers can be effectively reduced, and the homogeneity of the
library is improved.