The DNA methylome data generated using MeDIP-Seq technique from pig loin.

loin samples from four pairs of Duroc gilt littermates, with an average body weight of 101.6 ± 5.3 kg and average age of 184 ± 9 days, were collected from commercial breeding farms after slaughter.Genomic DNA was extracted from LDM samples and isolated using the phenol-chloroform-isoamyl alcohol method as follows: firstly, LDM tissue was frozen in liquid nitrogen, crushed with a mortar and pestle, and digested for 12 hr at 55 °C in a lysis buffer and 0.1 mg/ml of proteinase K. At the end of the incubation, 5 ml of protein PPT buffer was added to precipitate proteins without heating. After centrifugation of the solution at 13,000 rpm for 10 min, the supernatant was collected in a clean tube and 5 ml of phenol-chloroform-isoamyl alcohol (25:24:1) was added. After a further centrifugation at 13,000 rpm for 15 min, 5 ml of the aqueous phase was transferred to a clean tube, and the DNA was precipitated using 0.5 ml of 3 M sodium acetate and 10 ml of 100 % ethanol. The pellets were washed with 70 % ethanol, allowed to dry, and dissolved in TE buffer.For each sample, 1.2 μg of genomic DNA was randomly sheared to approximately 200–300 bp fragments using a Covaris S2. MeDIP libraries were constructed using the TruSeq DNA Sample Prep kit (Illumina, San Diego, CA, USA) following the manufacture’s recommended protocol. The DNA fragments were end-repaired, phosphorylated, poly(A)-tailed, and then ligated with Illumina single read adapters. The adapter-ligated DNA fragments sized approximately 250­–300 bp were screened by gel electrophoresis and purified with MinElute Gel Extraction kit (Qiagen, Valencia, CA, USA), and were used for MeDIP enrichment using Methylated DNA Immunoprecipitation kit (Diagenode, Liège, Belgium) following the manufacture’s recommendation. The immunoprecipitated DNA fragments were then PCR amplified: 98 °C for 30 sec; 10 cycles of 98 °C for 10 sec, 60 °C for 30 sec and 72 °C for 30 sec; and 72°C for 5 min. MeDIP libraries were quantified using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). Flow cells were prepared with 8 pM DNA using the manufacture’s recommended protocol and sequenced on an Illumina Genome Analyzer II to generate single-end 36 bp reads.

本研究从商品种猪场屠宰后的4对杜洛克后备母猪同窝同胞个体中采集背最长肌（longissimus dorsi muscle, LDM）样本，所有受试个体平均体重为101.6±5.3 kg，平均日龄为184±9天。研究采用酚-氯仿-异戊醇法从LDM样本中提取并分离基因组DNA，具体步骤如下：首先将LDM组织置于液氮中速冻，经研钵和研杵研磨破碎后，加入裂解缓冲液与0.1 mg/ml蛋白酶K，于55℃消化12小时。孵育结束后，加入5 ml蛋白质沉淀缓冲液（protein PPT buffer），无需加热即可沉淀蛋白质。将溶液以13000 rpm离心10分钟后，将上清液转移至洁净离心管中，加入5 ml体积比为25:24:1的酚-氯仿-异戊醇混合液。再次以13000 rpm离心15分钟后，取5 ml水相转移至洁净离心管中，通过加入0.5 ml 3 M乙酸钠与10 ml无水乙醇沉淀基因组DNA。所得DNA沉淀经70%乙醇洗涤后晾干，溶于TE缓冲液中。每份样本取1.2 μg基因组DNA，使用Covaris S2超声破碎仪将其随机打断为约200~300 bp的片段。参照试剂盒制造商的推荐方案，使用TruSeq DNA样本制备试剂盒（Illumina, San Diego, CA, USA）构建甲基化DNA免疫沉淀（Methylated DNA Immunoprecipitation, MeDIP）文库。首先对DNA片段进行末端修复、磷酸化修饰及poly(A)尾加尾，随后与Illumina单端测序接头进行连接。通过凝胶电泳筛选出长度约250~300 bp的接头连接产物，使用MinElute凝胶回收试剂盒（Qiagen, Valencia, CA, USA）纯化后，参照制造商推荐方案，使用甲基化DNA免疫沉淀试剂盒（Diagenode, Liège, Belgium）进行MeDIP富集。将免疫沉淀得到的DNA片段进行PCR扩增：98℃预变性30秒；随后进行10个循环的扩增反应（98℃变性10秒、60℃退火30秒、72℃延伸30秒）；最后72℃终延伸5分钟。使用Quant-iT PicoGreen dsDNA检测试剂盒（Invitrogen, Carlsbad, CA, USA）对构建好的MeDIP文库进行定量。参照制造商推荐方案，以8 pM的DNA浓度制备测序流动槽（flow cell），随后在Illumina Genome Analyzer II测序平台上进行测序，获得单端36 bp的读段数据。