Bisulfite Sequencing of GNAS and GNASXL Promoters

Bisulfite mutagenesis of genomic DNA was performed following the One-Step Modification Procedure of the Imprint DNA Modification Kit (Sigma). Methyl Primer Express Software (version 1.0, Applied Biosystems) was used to design primers that amplify regions proximal to the GNAS and GNASXL promoters. Both amplicons spanned SNPs identified by the whole genome sequencing of the B. t. indicus sire. Bisulfite PCR reactions were as previously reported.^12,13 (link) The cycling programs were as follows: initial denaturation at 94°C for 2 minutes and 15 seconds, 45 cycles of 94°C for 30 seconds, 56.1/58.7°C for 45 seconds (ramping rate: 1°C/second), and 72°C for 45 seconds, and final extension at 72°C for 5 minutes. The PCR products were resolved by agarose gel electrophoresis and isolated as previously described.¹² (link) The PCR amplicons were cloned using pGEM-T Easy Vector System (Promega) according to the manufacturer's instructions. Bacteria used were NEB 5-α F'I^q Competent E. coli (NEB). Bacteria colonies were screened by blue-white screen method and positive colonies were subject to Sanger sequencing as described above.