The experiment was carried out in triplicate. RNA libraries were prepared starting from 50 to 100 ng of total RNA from individual mice (n = 3 per group, three groups in total) and constructed using the Illumina TruSeq Stranded Small RNA Sequencing kit (Illumina Inc) according to the manufacturer’s instructions. Sequencing was performed at the Genomix platform (Sophia-Antipolis, France) using the HiSeq 2500 (Illumina Inc).

Read quality was assessed using FastQC and trimmed, against known common Illumina adapter/primer sequences, using trimmomatic. The SmallRNAs UCAGenomix pipeline with Illumina adaptor trimming was used, read sizes < 15 nucleotides were discarded. In order to describe the general distribution of sperm sncRNAs, trimmed reads kept were mapped to small RNA database using a recently developed annotation pipeline, SPORTS1.0 (Shi et al., 2018). We used the default settings and database files for the mouse genome GRCm38/mm10 that are available on the Sports github (https://github.com/junchaoshi/sports1.0). Averages summarized over biotypes were based on the default annotation result output files. Normalization of read abundance and differential expression analysis was performed by using DESeq R package on the Sports output files. The baseMean for each gene, the maximum of mean counts among all conditions, was at least 50 counts. Next generation sequencing (NGS) experiments have been deposited in the GEO Database with accession number (GSE138989).

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