DOI: 10.5061/dryad.ncjsxkt0m
title: Data for: Mouse sperm DNA-methylation changes induced by age and mechanistic target of rapamycin (mTOR) manipulation in Sertoli cells

description: This study was designed to study changes in sperm DNA methylation induced
 by age and by changes in activity of two complexes of the mechanistic
 target of rapamycin (mTOR) in Sertoli cells. Two transgenic mouse models
 were used. To inactivate mTOR complex one (mTORC1), we used Raptor
 (component of mTORC1) Sertoli-specific knockout model, and to inactivate
 mTOR complex two (mTORC2), we used Rictor (component of mTORC2)
 Sertoli-specific knockout model. Data was collected at two time-pints:
 postnatal week 8 and postnatal week 22. Sperm DNA methylation of all
 age/genotype groups was analyzed. Genes associated with differentially
 methylated regions were identified and biological categories enriched with
 these genes were identified.
description: To achieve Sertoli-specific knockouts, mice with floxed Rictor or
 Raptor (Rictor<sup>tm1.1Klg</sup>/SjmJ and
 B6.Cg-Rptor<sup>tm1.1Dmsa</sup>/J respectively) were crossed
 with mice with Cre recombinase controlled by the Amh Sertoli-specific
 promoter (129S.FVB-Tg(Amh-cre)8815Reb/J). All mice were purchased from
 Jackson Laboratories (Stock ##: 020649, 013188 and 007915 respectively).
 F3 wildtype and knockout male mice were euthanized at 8 or 22 weeks of age
 (n = 4 per genotype/timepoint). All procedures followed the guidelines of
 the National Institutes of Health Guide for the Care and Use of Laboratory
 Animals and the approval for this study was received from the
 Institutional Animal Care and Use Committee at University of
 Massachusetts, Amherst. At
 each euthanasia cauda epididymides were incised, cut three times, and
 incubated at 37C for 30 minutes in 1 ml of sperm wash buffer (Cat. #
 ART1006, Origio, Denmark). After incubation DNA extraction was done
 following the rapid method (1). Bisulfite conversion was performed on 100
 ng of genomic DNA using the EpiTect Fast DNA Bisulfite kit from Qiagen
 (Cat.# 59824) and DNA libraries were constructed using the NUGEN Ovation
 RRBS Methyl-seq System (Cat.# 0353) according to the manufacturer’s
 instructions. Libraries were sequenced on Illumina HiSeq 4000 at the Deep
 Sequencing Core Facility of the University of Massachusetts Medical School
 (Schrewsburry, MA) with an average of 22.7 million single end reads
 (50-bp) per sample. Raw
 reads were trimmed using TrimGalore and a NuGEN-specific adaptor trimming
 scripts available from GitHub (nugentechnologies/ NuMetRRBS). Trimmed
 reads were mapped using Bismark-Bowtie2 with no mismatch allowed.
 Methylation counts were called using Bismark extract. Differentially
 methylated regions were identified using the Methyl kit (v1.24.0) pipeline
 (2). In brief, the genome was tiled into sliding windows and a weighted
 methylation level was calculated for each window. We used a logistic
 regression model for p-value calculation subsequently adjusted for
 multiple comparison (FDR) using the SLIM method for final DMR
 identification. Individual DMRs were identified for a 100 bp sliding
 window with a minimum of 1 CpG. DMRs with methylation difference at FDR
 &lt; 0.05 were used to compare different age/genotype groups. The
 tiles identified in at least 3 samples out of 4 in an age/genotype group
 were included in comparisons. Each DMR was assigned to the closest gene
 (&lt;5 kb upstream transcription start site, promoter, 5’UTR, exon, or
 intron) or intergenic region using annotatr package (v1.24.0) (3) and
 mouse genome (mm10) assembly data from ENSEMBL. We used Metascape (4) to
 analyze biological categories associated with differentially methylated
 regions. References (1)       Wu, H,
 et al. Rapid Method for the Isolation of Mammalian Sperm DNA.
 BioTechniques 2015, 58 (6). (2)       Akalin, A, et al.
 MethylKit: A Comprehensive R Package for the Analysis of Genome-Wide DNA
 Methylation Profiles. Genome Biol. 2012, 13 (10), R87.
 (3)       R. G. Cavalcante, M. A. Sartor, annotatr: genomic
 regions in context. Bioinforma. Oxf. Engl. 33, 2381–2383 (2017)
 (3). (4).      Zhou, Y, et al. Metascape Provides a
 Biologist-Oriented Resource for the Analysis of Systems-Level Datasets.
 Nat. Commun. 2019, 10 (1).
description: The dataset contains the following
 files: 1.     Fastq files for individual RRBS-seq
 experiments (individual mice) as follows (individual mice ID are included
 in file names): Rictor KO, postnatal week 8 – samples
 M8, M21, M22, and M90 Rictor WT, postnatal week 8 –
 samples M10, M11, M12, M20 Rictor KO, postnatal week 22
 – samples M32, M33, M39, M82 Rictor WT, postnatal week
 22 – samples 2i, M19, M38, M81 Raptor KO, postnatal
 week 8 – samples A1, A2, B1, B20 Raptor WT, postnatal
 week 8 – samples A10, A20, A22, B21 Raptor KO,
 postnatal week 22 – samples E22, F1, F10, G2 Raptor WT,
 postnatal week 22 – samples E20, F2, F20, G1 2.     MS
 Excel files containing annotated differentially methylated DNA regions
 form the following comparisons: WT22_vs_WT8_mTORC1.xlsx
 – comparison of sperm DNA methylation in 8 and 22 weeks old wildtype mice
 in mTORC1 experiment. KO22_vs_WT22_mTORC1.xlsx –
 comparison of sperm DNA methylation in Raptor knockout and wildtype 22
 weeks old mice. WT22_vs_WT8_mTORC2.xlsx – comparison of
 sperm DNA methylation in 8 and 22 weeks old wildtype mice in mTORC1
 experiment. KO8_vs_WT8_mTORC2.xlsx – comparison of
 sperm DNA methylation in Rictor knockout and wildtype 8 weeks old
 mice. 3.     MS Excel files containing results of
 Metascape enrichment analysis for genes associated with significant DMRs
 identified in the following comparisons:
 Enrichment_WT22_vs_WT8_mTORC1.xlsx – enrichment for genes
 associated with DMRs identified in the comparison 8 and 22 weeks old
 wildtype mice in mTORC1 experiment.
 Enrichment_KO22_vs_WT22_mTORC1.xlsx – enrichment for genes
 associated with DMRs identified in the comparison of Raptor knockout and
 wildtype 22 weeks old mice.
 Enrichment_WT22_vs_WT8_mTORC2.xlsx – enrichment for genes
 associated with DMRs identified in the comparison 8 and 22 weeks old
 wildtype mice in mTORC2 experiment.
 Enrichment_KO8_vs_WT8_mTORC2.xlsx – enrichment for genes
 associated with DMRs identified in the comparison of Rictor knockout and
 wildtype 8 weeks old mice.