RNA library preparation was performed as described in the Illumina TruSeq Stranded Total RNA LT Sample Preparation Kit with Ribo-Zero Gold (Illumina Inc.). Briefly, ribosomal RNA was depleted from 0.3 to 1 μg of total RNA. Following depletion, mRNA was fragmented to an average size of 200 to 400 bp at 94°C for 4 min. The cleaved RNA fragments were copied into first-strand cDNA using a reverse transcriptase (Invitrogen) and random primers. The first-strand cDNA was converted into double-stranded DNA in the presence of deoxyuridine triphosphate (dUTP). The incorporation of dUTP in second-strand synthesis quenched the second strand during amplification and thus improved the strand specificity of the library. These cDNA fragments then had the addition of a single “A” base and subsequent ligation of the adapter. The products were purified and enriched with PCR to create the final library. After qualification with the Agilent 2100 Bioanalyzer (Agilent Technologies) with the DNA chip and quantification with the Qubit 3.0 Fluorometer (Invitrogen), the samples were subjected to 150-bp paired-end sequencing using an Illumina HiSeq X Ten platform (Illumina Inc.). The raw data were subjected to quality control analyses using Seqtk. The RNA-seq data were then mapped to the human reference genome hg19 with TopHat2 (version: 2.0.9). The HERVs were obtained from RepeatMasker (www.repeatmasker.org/species/hg.html) for expression analyses by HTSeq-count. In total, 553 HERV species (distributed across 523,656 genomic loci) were included. The HERV differential analysis was performed using edgeR.

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