MSCC Library Construction Protocol

The MSCC libraries were constructed according to the description of Guo et al. [21 (link)] with few alterations. For each of the five samples (S0h, S4h, S12h, S24h and S96h), two libraries were constructed. Two custom adaptors that contained a 5′ CG overhang and 3′ NN overhang, respectively, were created. For the HpaII library, 2 μg of genomic DNA combined with standard DNA was digested with HpaII (New England Biolabs [NEB]) for 2 h. Adaptor A was ligated to the resulting fragments. The reaction products were then incubated with Bst DNA polymerase (NEB) for 20 min. After digestion with MmeI (NEB), adaptor B was added to the reaction mixture incubated with T4 DNA ligase (NEB) overnight. The products were purified with Agencount AMPure XP Beads (Beckman) and then run on a 2% E-Gel® EX Gel (Invitrogen). The target band at approximately 140 bp was purified with the QIAquick Gel Extraction Kit (Qiagen). An 8-cycle PCR protocol was performed on the purification products. For the inverse library, after HpaII digestion in the first step, the fragmented ends were deactivated by incubation with Antarctic Phosphatase (NEB). The products were digested with MspI and then treated with the same procedure as the HpaII library.