# Placental Transcriptome Adapations to Maternal Nutrient Restriction in Sheep
## 4 Materials and Methods
### RNA Extraction, Sequencing, and Analyses  
Total RNA was extracted from snap-frozen placentomes using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's recommendations. Extracted RNA was treated with DNase I (Qiagen, Hilden, Germany) and purified using a RNeasy Mini Kit (Qiagen), before the RNA was quantified and its quality was assessed using a NanoDrop and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA),respectively. An RNA integrity number (RIN) of >8 and a 260/230 value of >1.8 were considered acceptable. Extracted RNA was stored at \(-\)80 \({}^{\circ}\)C until further analyses.  
Total RNA from the samples was submitted to the University of Missouri DNA Core facility (Columbia, Missouri, USA). Library construction and sequencing was conducted following the manufacturer's protocol with reagents supplied in Illumina's TruSeq Stranded mRNA sample preparation kit. Libraries were multiplexed and sequenced from both directions as 75 base pair paired-end reads on one lane on a NextSeq500 instrument. The raw sequences (FASTQ) were subjected to quality check via FastQC ([[http://www.bioinformatics.babraham.ac.uk/projects/fastqc/](http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)]([http://www.bioinformatics.babraham.ac.uk/projects/fastqc/](http://www.bioinformatics.babraham.ac.uk/projects/fastqc/)) (accessed on 16 July 2017)). The program fattrim ([[https://ccb.jhu.edu/software/fattrim/](https://ccb.jhu.edu/software/fattrim/)]([https://ccb.jhu.edu/software/fattrim/](https://ccb.jhu.edu/software/fattrim/)) (accessed on 16 July 2017)) was used to remove adapters, perform quality trimming (phred score > 30) by a sliding window scan (6 nucleotides), and select read lengths of 30 nucleotides or longer after trimming. The reads obtained from the quality control step were mapped to the sheep reference genome (Oar_v3.1) using Hisat2 aligner [50]. The program FeatureCounts [51] was used to quantify read counts by using the sequence alignment files of each sample. Genes with evidence of expression (counts per million; rowSum > 5) were used for model-based differential expression analysis using the edgeR robust method [52]. The false discovery rate (FDR) \(\leq\) 0.10 was used as threshold for statistically significant differential expression of genes. Only protein-coding genes were included. Venn diagrams were produced using Venny 2.1 ([[http://bioinfogp.cnb.csic.es/tools/venny/index.html](http://bioinfogp.cnb.csic.es/tools/venny/index.html)]([http://bioinfogp.cnb.csic.es/tools/venny/index.html](http://bioinfogp.cnb.csic.es/tools/venny/index.html)) (accessed on 13 May 2021)). Over-representation analyses using DEG lists were conducted using ToppFun ([[http://toppgene.cchmc.org/](http://toppgene.cchmc.org/)]([http://toppgene.cchmc.org/](http://toppgene.cchmc.org/)) accessed on 18 May 2021)) with default settings [53] to identify Gene Ontology (GO) terms for the molecular function ontology (FDR < 0.05). Sheep Ensembl IDs were converted to human Ensembl IDs (genome assembly GRCh38.p13) in order to facilitate pathway analyses. Enrichment analyses were performed using gene set enrichment analysis software v4.1.0 ([[http://gsea-msigdb.org/](http://gsea-msigdb.org/)]([http://gsea-msigdb.org/](http://gsea-msigdb.org/)) (accessed on 14 May 2021)) [54], with statistical significance set at an FDR \(q\)-value < 0.25. Transcription factors were identified using the Tf2 DNA database ([[http://fiserlab.org/tf2](http://fiserlab.org/tf2) dna_db/]([http://fiserlab.org/tf2](http://fiserlab.org/tf2) dna_db/) (accessed on 18 May 2021)). KEGG pathway analyses were performed using the pathview package in R [55]. Data files were deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) under accession number GSE180182.