Nebnext multiplex oligo

CMS-IP was performed similar to pervious described (Huang et al., 2012 (link); Pastor et al., 2011 (link)). Briefly, genomic DNA from in vitro cultured pro-B cells was isolated with PureLink Genomic DNA kit (Thermo Fisher) and were spiked-in with cl857 Sam7 λDNA (Promega,Madison, WI) and PCR-generated, hmC-containing puromycin-resistant gene at a ratio of 200:1 and 100,000:1, respectively. DNA was sheared with a Covaris E220 (Covaris), end-repaired, A-tailed, ligated with methylated Illumina adaptors (NEB, Ipswich, MA), and bisulfite-converted (MethylCode Bisulfite Conversion Kit, Thermo Fisher). Bisulfite-converted DNA was denatured and immunoprecipitated with anti-CMS serum. Immunoprecipitated DNA was PCR-amplified with barcoded primers (NEBNext Multiplex Oligos for Illumina, NEB) for 15 cycles with Kapa HiFi Uracil+ (Kapa Biosystems, Wilmington, MA). Resulting libraries were sequenced with a HiSeq2500 system for 50 bp paired-end reads (Illuminia, San Diego, CA). The sequence reads were mapped to mm9 with Bismap, and CMS-enriched genomic regions were identified using the ‘findPeaks' command in HOMER with the 'histone’ mode and default parameters (Heinz et al., 2010 (link)).

RNA libraries, with equal RNA amounts, from five curly-coated Mangalitza, seven Mangalitza×Angeln Saddleback shoats, and seven straight-coated individuals (see Supplementary Table S1) were prepared using TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, United States) and indexed by NEBNext Multiplex Oligos for Illumina (Unique Dual Index Primer Pairs, NEB, Ipswich, United Kingdom). The prepared libraries were set in equimolar dilutions and submitted for sequencing for 66 million reads. In total, 32 libraries were sequenced on an Illumina NextSeq500 for 2 × 75 bp, further 15 libraries were sequenced on an Illumina NovaSeq6000 for 2 × 100 bp. In the next step, the binary base call (BCL) sequence output files were converted to FASTQ format and underwent quality control using FastQC version 0.11.8 (Andrews, 2010 ). FASTQ files were further processed using fastp version 0.20.0 (Chen et al., 2018 (link)) to control for adaptor content and discarding long stretches of N base >50 (--n_base_limit 50), 70% unqualified bases (unqualified_percent_limit 70), 1% complexity threshold (complexity_threshold 1) and low-quality reads (link)) based on Sus Scrofa11.1 genome reference (Ensembl, database version 101.111). In addition, STAR quantMode was run to extract raw gene counts.