RAD-Seq of mus musculus: male feces
DNA ( 1 pg-200 ng) was digested with 4 U of the enzyme BcgI (NEB) for 3 h at 37 . Subsequently, the adaptors were ligated to the DNA fragments. The ligation reaction was performed by combining 5 l of digested DNA with 10 l of a ligation master mix containing 0.2 M each of two adaptors and 800 U T4 DNA ligase (NEB). Ligation was carried out at 4  for 12 h. Then, ligation products were amplified, and PCR products were subjected to 8% polyacrylamide gel. Bands of approximately 100 bp were excised from the polyacrylamide gel, and the DNA was diffused from the gel in nuclease-free water for 12 h at 4 . Sample-specific barcodes were introduced by PCR with platform-specific barcode-bearing primers. Each 20 l PCR contained 25 ng of gel-extracted PCR product, 0.2 M of each primer, 0.3 mM dNTP, 1Phusion HF buffer and 0.4 U Phusion high-fidelity DNA polymerase (NEB). PCR products were purified using QIAquick PCR purification kit (Qiagen) and then subjected to sequencing using the Illumina Nova PE156 platform.