PC-Testis3
Through the interaction of 3'-5' exonuclease and polymerase, DNA fragments with protruding ends were repaired. A single "A" base is introduced at the 3' end of the repaired flat DNA fragment, and the 3' end of the connector contains a single "T" base to ensure that the DNA fragment and the connector can be connected through "A" and "T" complementary pairing and prevent the DNA insert fragments from annealing with each other during this process. Under the action of ligase, the labeled splices are incubated to connect with DNA fragments. VAHTS DNA clean beads were used to purify the free splices and segments without splices. PCR was used to selectively enrich DNA fragments with connectors at both ends and amplify the DNA library with fewer cycles to avoid errors in PCR amplification. An Agilent 2100 was used to control the quality of the PCR-enriched fragments and verify the size and distribution of DNA library fragments. Multiplexed DNA libraries were homogenized to 10 nM and then mixed in equal volumes. The mixed library (10 nM) was diluted to 4~55 pM and sequenced. Next-generation sequencing (NGS) was used to perform paired-end sequencing on these libraries (paired-end, PE) based on the Illumina sequencing platform.