HTGTS libraries were constructed as described (Dong et al., 2015 (link)). Briefly, genomic DNA of CH12F3 or its mutants were extracted after stimulation for 3 days. The genomic DNA was sonicated and amplified by LAM-PCR with 5′ Sμ biotin primer (5′-CAGACCTGGGAATGTATGGT-3′). The Biotinylated products of PCR were captured by Dynabeads MyOne streptavidin C1 beads (Invitrogen), ligated with bridge adapters on-bead. The ligated products were amplified by second-PCR to add adaptor. Then, the products of PCR were blocking with endonuclease Afill to remove germline genomic DNA fragment. The third round PCR was performed to add Illumina Miseq-compatible adapters to conduct MiSeq sequencing. The HTGTS data were analyzed as described (Dong et al., 2015 (link); Panchakshari et al., 2018 (link)). Data were presented as mean ± SEM (Student’s t-test, *p < 0.05, **p < 0.01, ***p < 0.001).
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