DNA methylation in sperm of rats at two ages exposed or not to 2,2',4,4'-tetrabromodiphenyl ether

Introduction

This study was designed to determine the potential of environmentally relevant levels of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) to induce age-dependent changes in a rat’s sperm epigenome. The methods used to generate the files are described below.

Experimental design

Twelve seven-week-old pregnant Wistar rats were divided into two groups (6 per group) – control and BDE-47 exposed group. Between pregnancy day 8 and postnatal day 21 (PND21), dams in each group were fed from the tip of pipette 0.2 µL/g body weight of the vehicle (tocopherol-stripped corn oil) or the same volume of a 1 mg/mL solution of BDE-47 daily. The BDE-47 group resulted in an exposure level of 0.2 mg/kg body weight of BDE-47 per day. On PND65 and PND120, one male pup randomly selected from each litter was euthanized, and epididymal motile spermatozoa were collected via the swim-up procedure as described in detail elsewhere (Suvorov et al., 2018). Sperm DNA was extracted using the rapid method (Wu et al., 2015). Extracted sperm DNA was subjected to reduced representation bisulfite sequencing (RRBS).

Reduced representation bisulfite sequencing

For RRBS, bisulfite-converted libraries were prepared from 100 ng of the sperm DNA using Ovation RRBS Methyl-Seq System and EpiTect Fast DNA Bisulfite Kit (Cat. #59824, Qiagen) following manufacturers’ protocols. Sequencing of libraries was done using the HiSeq 2500 sequencing system (Illumina) in Deep Sequencing Core Facility of the University of Massachusetts Medical School (Schrewsbury, MA) with an average of 18.0 million unique reads per sample.

Bioinformatic analysis

Raw reads from the sequence were processed following the recommended protocol for libraries prepared with Ovation RRBS Methyl-Seq System (NuGEN) and then mapped to the rn6 Rattus norvegicus reference genome using Bismark (version 0.16.1) and bowtie-2 (version 2.2.9). PCR duplicates were removed using nudup.py (version 2.2). The resulting SAM files for the control and BDE-47 group for the two ages, PND65 and 120 are uploaded.