RNA-seq of Bos taurus_H3
Total RNA was extracted, and mRNA was purified using polyT oligo-attached magnetic beads. cDNA libraries were prepared using an NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswitch, MA, USA). After cluster generation, the libraries were sequenced on an Illumina NovaSeq platform, and 150-bp paired-end reads were generated. Raw reads in fastq format were processed using fastp software (version 0.1), and clean data were obtained by excluding reads containing adapters or poly-Ns and low-quality reads. Clean reads were mapped to the Bos taurus reference genome in Ensembl_(ftp://ftp.ensembl.org/pub/release-106/gtf/bos_taurus/) using Hisat2. The read numbers mapped to each gene were counted using featureCounts (version 1.5.0-p3), and the read count was converted to reads per kilobase of transcript per million (RPKM) mapped reads. Differential expression analysis of H and NH groups was performed using the DESeq2 R package (1.20.0).25 Genes with fold change > 1 and P < 0.05 were assigned as differentially expressed. Translational efficiency (TE) was calculated as the ratio of RPKM in Ribo-seq to RPKM in RNA-seq. Differential TE was analyzed by RiboDiff (version 0.2.1).