RNA sequencing data of mouse 2-cell embryos treated with DMSO

To understand the effect of DMSO in preimplantation embryos, we have treated mouse 1 cell zygotes with DMSO and found that DMSO treatment caused 2 or 4 cell embryonic arrest and altered the acetylation levels of mouse preimplantation embryos To illustrate the mechanism of DMSO in mouse preimplantation embryos, fertilized zygotes have been treated with 2% of DMSO and then performed RNA-seq analyses. Differentially expressed genes were identified using DESeq2 after adjustment for false discovery rate (FDR q value < 0.05). Gene Set Enrichment Analysis (GSEA) was also performed to identify biological pathways significantly modulated by DMSO. Raw and processed RNA-seq data were deposited and made publicly available on the Gene Expression Omnibus (GEO; GSE124598). The data presented in this article are related to the research paper entitled “DMSO impairs the transcriptional program for maternal-to-embryonic transition by altering histone acetylation”, available in Biomaterials [1].


Data
Datasets presented here were employed in the main work "DMSO impairs the transcriptional program for maternal-to-embryonic transition by altering histone acetylation" Kang et al., 2020 [1]. Fig. 1 illustrates the experimental procedure. RNA-seq analysis was performed in 2-cell mouse embryos cultured after supplementation of 2% DMSO. The raw data generated from Illumina sequencing were deposited on the Gene Expression Omnibus (GEO) with the reference number GSE124598 (https:// www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE124598).
RNA-seq analysis was performed in the 2-cell embryos with/without DMSO supplementation. In total, 3,742, which is~20.29% of the total valid genes, genes were differentially expressed in DMSOtreated embryo compared with control embryo with criteria of FDR < 0.05. Of these differentially expressed genes, 1,415 genes were up-regulated, whereas 1,758 genes were down-regulated in DMSOtreated embryo (Fig. 2). DEGs were significantly enriched in total 72 KEGG and REACTOME pathways terms (adjusted p-value < 0.01) and the terms were mainly clustered into 4 characterized groups (Fig. 3).
Next, we interpreted potential interactive pathways among DEGs associated with epigenetic gene expression, histone modifications (acetylation and methylation) in DMSO treated group using cerebral Specifications Table   Subject Developmental

Value of the Data
The network data analysis such as gene ontology (GO), molecular pathways, and transcriptomic analysis of 2-cell embryos treated with DMSO could provide novel insights about the differential responses between maternal and embryonic clock. Mapped reads data and TPM values in raw data could be useful to predict developmental arrest of early embryos via incomplete epigenetic reprogramming and cellular stress induced by DMSO. RNA-seq analysis offer researchers to test whether DMSO is associated with possible toxicity and/or a range of serious side effects in cellular function and growth. Mouse preimplantation embryo-based assays can provide timely alerts about widespread applications of DMSO as a positive control or drug solvent agent. layout ( Fig. 4). Most of DEGs for histone modifications and binding events are significantly depressed at specific and highly characteristic genomic elements and locations in DMSO-treated groups, indicating that DMSO exhibits specific regulatory mechanisms related to regulation of transcription factors, compared with control embryos.
In this study, we proved our hypothesis by RNA-seq analysis to monitor the early embryonic impacts after exposure to DMSO and identified previously unknown underlying molecular mechanisms that explain the DMSO-induced embryonic toxicity, embryo loss, and infertility. Our study suggests for the first time that DMSO exposure induces a significant alteration in gene expression and the functionality of preimplantation embryos via alternations in epigenetic reprogramming. Thus, our findings  emphasize that the use of DMSO as a standard control test or solvent requires far more cautious consideration, because DMSO can alter cell function by acting as a proteasome or HDAC inhibitor as well as inducing cell toxicity.

Animals and embryo collection
BDF1 (C57BL/6 Â DBA/2; F1; Orient Bio Co. Ltd) mice (8e12 weeks olds) were used for analysis according to guidelines approved by the committee on animal care and use at Konkuk University (IACUC approval number: KU18199). Intraperitoneally injection was carried out in female mice were with pregnant mare's serum gonadotropin (PMSG; G4527, Sigma Aldrich; 5IU) followed human Fig. 3. Interactive string network of KEGG and REACTOME pathways for DEGs. Enriched KEGG and REACTOME pathways for DEGs are mainly clustered as Group 1e4 using ClueGO plug-in in Cytoscape 3.6. Detailed genes on each pathway node are listed in Table 1 and Supplementary Data. chorionic gonadotropin (hCG; CG10, Sigma Aldrich; 5IU) 48 h later, then mated with male mice. Fertilized oocytes with two pronuclei were collected from oviduct at 18e20 h of post hCG injection and each 10 zygotes was cultured in 20ul KSOM (95mM NaCl, 2.5mM KCl, 0.35mM KH 2 PO 4 , 0.2mM MgSO 4 , 10mM Sodium Lactate, 0.2mM Glucose, 0.2mM Sodium pyruvate, 25mM NaHCO 3 , 1mM Glutamine, 0.01mM Ethylenediaminetetraacetic acid, 5mg/ml Bovine albumine serum) supplemented with 2% DMSO (D2650, Sigma Aldrich) or without. BDF1 embryos with second polar body were collected and cultured in KSOM with/without 2% DMSO for further analysis.

Library preparation and RNA-seq
Fifty 2-cell embryos from each control and DMSO-treated group were directly subjected to cDNA synthesis using SMARTer® Ultra® Low Input RNA Kit (634940, Clonetech) according to the manufacturer's instructions. RNA quality was determined using the Agilent Bioanalyzer High Sensitivity DNA kit (5067-4626, Agilent). The synthesized cDNAs with 150-200bp size were used for the preparation of sequencing library using Low Input DNA Library Prep Kit (634946, Clonetech) according to the manufacturer's instructions, and subjected to size selection, followed paired-end reads data were obtained by performing 50 bp sequencing using HiSeq2500 (Illumina).

RNA-seq data analysis
Reads were pseudomapped using kallisto [2] with default parameters by transcriptome index from FASTA formatted transcriptomes files (GRCm38.re179) of ENSEMBL transcript database (mm10). Transcript abundance of each gene was quantified with the parameters (quant -t -b 100) as transcripts per kilobase million (TPM) using kallisto. Gene-scaled TPM values for each gene transcript were summed by tximport [3] in R/Bioconductor. Differentially expressed gene (DEG) were analyzed by Based on GO enrichment test by ClueGO, heat maps and pathway-like visualizations for DEGs that associated with epigenetic gene expression (A), histone methylation (B) and histone acetylation (C) were created using CluePedia plug-in in Cytoscape 3.6 software. Functional relations between DEGs were drawn by colored lines, which represent activation (green), catalysis (purple), inhibition (red), protein modification (light purple) and reaction (black). DESeq2 [4] in R/Bioconductor with the parameters (baseMean counts >14; false discovery rate (FDR) < 0.05).

Pathway enrichment test and in silico analysis
DEGs were tested for pathway enrichment score in KEGG and REACTOME pathways using ClueGO [5] plug-in in Cytoscape 3.6 (http://www.cytoscape.org). To search potential associations among DEGs specific gene ontology (GO) terms regarding epigenetic gene expression, histone acetylation and histone methylation, ClueGO enrichment test were integrated into CluePedia [6] plug-in in Cytoscape 3. 6 and analyzed.