Hisseq

Briefly, genomic DNA was isolated from flash frozen muscular tissue. Then, the construction of RRBS libraries and paired-end sequencing using Illumina HisSeq analyzer was performed at Novogene technology co., LTD. (Beijing, China). Raw sequencing data were processed by an Illumina base-calling pipeline. Clean reads were aligned to the pig reference genome (Sscrofa11.1) using Bismark (Felix and Andrews, 2011 (link)) after removing adaptor sequences. Next, ML were measured using bismark_methylation_extrator program. A quality control procedure was adopted to ensure the high data quality by 1) retaining only CpG cytosines across all samples; 2) removing CpG sites with missing methylation values at >35 samples; 3) removing CpG sites with coverage <10 reads within a sample. A total of 3,083,713 CpG sites were retained for further analysis. The distribution of CpG sites were conducted using R package Rldeogram v.2.2 (Hao et al., 2020 (link)).

Briefly, genomic DNA was isolated from flash frozen muscular tissue. Then, the construction of RRBS libraries and paired-end sequencing using Illumina HisSeq analyzer was performed at Novogene technology co., LTD (Beijing, China). Raw sequencing data were processed by an Illumina base-calling pipeline. Genomic DNA was digested with MspI enzyme at 37 °C for 16 h. The DNA fragments after enzyme digestion were repaired at the end, and the sequencing adapters with all cytosine methylated were attached. The inserted DNA fragments with the length ranging from 40 to 220 bp were selected for glue cutting. Then, Bisulfite conversion was carried out. After that, the unmethylated C was changed to U (after PCR amplification to T), while the methylated C remained unchanged. Finally, PCR amplification was carried out to obtain the final DNA library. Clean reads were obtained from the raw data after removing reads containing adaptor sequences, unknown, or low-quality bases. The process of quality control was carried out using Trimmomatic software [41 (link)]. Quality control was adopted to access the high data quality by (1) removing low-quality reads using a sliding window method (SLIDINGWINDOW: 4:15); (2) removing reads including adaptor sequences (ILLUMINACLIP: adapter.fa: 2:30:7:1: true); (3) removing reads with tail quality lower than 3 or with unknown bases (TRAILING: 3).