2.2.1 Prevention of CGat adapters’ self-ligation
The self-ligation of CGAT adapters were tested in two groups, the adapters with T4 ligase alone, and adapters with the combination of T4 ligase and ClaI enzymes. The T4 DNA ligase group contains 200 ng of CGat adapters, 5 μL of 10X CutSmart™ buffer , ATP (10 μM) 5 μL, T4 DNA Ligase (New England Biolabs (Beijing) LTD)1 μL(400uints) (last added) in a 50 μL reaction system. The components in ClaI group is the same as the T4 ligase control group except for the addition of Cla I restriction enzyme(New England Biolabs (Beijing) LTD) 2.5 μL (25uints). The reactions were performed overnight in a 37-degree water bath. After the reaction completed, 5 μL of the reaction products was visualized by electrophoresis under a 2.5% agarose gel under 100 V 30 min.
The ClaI was further tested in promoting the formation of CGat adapters and insert ligation. As tabulated in Table 2, the effect of ClaI was evaluated in the groups 4 and 5, one without and one with ClaI. The restriction enzyme HpaII was used to digest the artificial template and added in groups 2, 4, and 5.
The ligation product of the insert and adapters was then used as template for PCR amplification. The primer sequence is: 5-ccatccagcatccaactcggtgggggtggggat-3,5-gtcagctccacgcatacattacg-3. PCR reaction system: 2× taq Master Mix (Vazyme Biotech Co.,Ltd)10 μL, junction primer 1 μL, destination fragment primer 1 μL, ligation product 3 μL, replenish water to a total volume of 20 μL. PCR was set with 95 °C 2min 1cycle, 95℃ 10s,58℃ 20s,72℃ 30s,20 cycle, and a final cycle at 72℃ 1min. The PCR product was sent for Sanger’s sequencing.