DOI: 10.5281/zenodo.10119321
title: Long-term changes in pituitary gene expression following developmental exposure to environmental contaminants (BPS, BDE-47, or TBBPA) in male mice.

description: Experimental Design
In this study we evaluate the long-term gene expression changes in pituitary in male mice exposed developmentally to one of three known endocrine disrupting chemicals: bisphenol-S (BPS), 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), and 3,3',5,5'-tetrabromobisphenol A (TBBPA).  Male mice were exposed to chemical treatment through their mothers' during pregnancy (umbilical blood flow) and nursing, from pregnancy day 8 through weaning at postnatal day 21 (PND21). Each chemical exposure was calculated to equal 0.2mg/kg bw/day. The details of exposure protocol are described elsewhere (Kim et al, 2015). The male pups were allowed to grow untreated until adulthood at PND140. At PND140, male mice were euthanized, then their pituitaries removed, snap frozen in liquid nitrogen, and then stored at -80°C. 
The data-files described below represent major steps of our analysis:
1. FASTQ files for mouse pituitary RNA-seq data.
Male mouse pituitary RNA was isolated using a Trizol protocol, checked for purity and concentration and then processed for mRNA sequencing using the Illumina TruSeq kit and protocol (TruSeq Stranded mRNA LP, Cat # 20020594 and TruSeq RNA Sg Idx SetB, Cat # 20020493, Illumina, San Diego, CA) following the manufacturer's recommended procedures. High throughput sequencing was conducted using the NextSeq500 sequencing system. cDNA libraries were single-end sequenced in 76 cycles using a NSQ 500/550 Hi Output KT v2.5 (Cat #20024906 Illumina, San-Diego, CA) in one multiplex run (N=3/exposure group).  Read filtering, trimming, and de-multiplexing were performed using the BaseSpace cloud service by Illumina (https://basespace.illumina.com/home/index, RRID:SCR_011881). Processed reads were mapped to the mouse reference genome (MM10) using the RNA-Seq Alignment v. 1.1.1. software with Bowtie 2. Each FASTQ file is a compressed file representing data from one sequencing flow cell lane for each sample (4 files per sample). Sample identifiers are coded for treatment: X = Vehicle control, R = TBBPA, C = BDE-47, E = BPS. 
2. Differential expression data.
Aligned reads were used to assemble transcripts and analyze differential expression using Cufflinks Assembly & DE v. 2.1.0. package. Reads aligned to known annotated regions for both control and exposed groups were used to calculate log2 FPKM ratios.  Differentially expressed genes were identified as genes altered with false discovery rate significance ≤ 0.05 (FDR, q ≤ 0.05).  Data on all exposure groups are shown in different sheets of the same file - Differential_expression.xlsx.
3. Enrichment of biological categories associated with DEGs induced by chemical exposures. 
All differentially expressed genes were uploaded to Metascape for the analysis of enriched biological categories using default settings. Results of Metascape analysis are shown in two MS Excel files per exposure group, one showing negatively enriched categories and one positively enriched categories. The title of each file consists of three parts connected via underscore sign: the name of the chemical, the direction of enrichment, and the name of analysis - metascape (e.g., BDE-47_negative_metascape.xlsx).
4. Pathway analysis for chemical exposures.
All differentially expressed genes were uploaded to Ingenuity Pathway Analysis and enriched canonical pathways were identified  using default settings . Altered molecular or disease pathways, their p-values, and associated differentially expressed genes are provided for each exposure. Data for all exposure groups are shown in different sheets of the same file - IPA_pathway_analysis.xlsx.
References:
Kim B, Colon E, Chawla S, Vandenberg LN, Suvorov A. Endocrine disruptors alter social behaviors and indirectly influence social hierarchies via changes in body weight. Environ Health. 2015 Aug 5;14:64. doi: 10.1186/s12940-015-0051-6. PMID: 26242739; PMCID: PMC4524022.