Equine Cervical Remodelling during Prepartum Period and Placentitis: A Transcriptomic Approach (Mucosa)

Placentitis was induced in five mares at approximately 290d of gestation (placentitis group), four mares with gestationally age-matched (290 d) pregnancies did not receive any treatment (control group), and the remaining three mares were maintained until approximately 330 d of gestation (prepartum group). For induction of placentitis in the former group, Streptococcus equi subsp. zooepidemicus was introduced intracervically. Cervical mucosa samples were collected from mares with experimentally induced placentitis (290 d GA, n =5), normal pregnant mares (290 d GA, n=4), and prepartum mares (330d GA, n=3). Total RNA was isolated from all samples using RNeasy Mini Kit (#74104; Qiagen), and DNA digestion was performed on-column using RNase-free DNase I (#79254: Qiagen), followed by cleanup procedures. Paired-end reads with 150 nucleotides in length were produced. A TruSeq Stranded mRNA library prep kit (Illumina, San Diego, CA) was used to prepare libraries for mRNA sequencing. Libraries were loaded onto an Agilent DNA 1000 chip and validated on an Agilent 2100 Bioanalyzer (Agilent). Quantitation was performed with the Illumina Library Quantification Kit, ABI Prism qPCR Mix from Kapa Biosystems. Libraries were run on a NextSeq 500 v2 (Illumina) 300cycles High Output kit in a 2x150 base pairs with paired-end reads. The Fastq files were evaluated for read quality using FastQC 0.11.4. Subsequently, Trim Galore 0.4.1 was used for the adapter and read quality trimming (Phred score threshold of 30). Reads were mapped to the Equus caballus reference genome (EquCab 3.0) using the software STAR 2.5.3a, then annotated with the equine reference annotation from NCBI using Cufflinks 2.2.1. Fragments per kilobase per million (FPKM) was used to determine the expression level of genes. Lastly, we used Cuffdiff 2.2.1 to calculate differentially expressed genes (DEG) between samples from the control and urea groups. The significance level was set at the FDR-adjusted p-value of the test statistic < 0.05 using the Benjamini-Hochberg correction.

本实验对5匹妊娠约290天的母马诱导胎盘炎（胎盘炎组）；另设4匹妊娠龄匹配（290天）的妊娠母马作为对照组，不施加任何处理；剩余3匹母马饲养至妊娠约330天，设为产前组。胎盘炎组的胎盘炎诱导采用宫颈内接种马链球菌兽疫亚种（Streptococcus equi subsp. zooepidemicus）。分别从实验性胎盘炎母马（290天妊娠龄，n=5）、正常妊娠母马（290天妊娠龄，n=4）及产前母马（330天妊娠龄，n=3）采集宫颈黏膜样本。所有样本均采用RNeasy Mini Kit（货号#74104；Qiagen）提取总RNA，随后使用无RNase的DNase I（货号#79254；Qiagen）进行柱上DNA消化，并完成后续纯化步骤。生成长度为150核苷酸的双端测序读段；采用TruSeq Stranded mRNA文库制备试剂盒（Illumina，美国加利福尼亚州圣地亚哥）构建mRNA测序文库。将文库加载至Agilent DNA 1000芯片，通过Agilent 2100生物分析仪进行文库质检。使用Illumina文库定量试剂盒及Kapa Biosystems的ABI Prism qPCR Mix完成文库定量。将文库置于NextSeq 500 v2（Illumina）平台，采用300循环高通量试剂盒进行2×150bp双端测序。使用FastQC 0.11.4评估Fastq文件的测序读段质量，随后通过Trim Galore 0.4.1完成接头序列及测序质量修剪（Phred质量阈值设定为30）。采用STAR 2.5.3a软件将测序读段比对至马（Equus caballus）参考基因组（EquCab 3.0），再通过Cufflinks 2.2.1结合NCBI提供的马参考注释文件完成基因注释。采用每百万reads每千碱基片段数（FPKM, Fragments per kilobase per million）计算基因表达水平。最后使用Cuffdiff 2.2.1计算对照组与尿素组样本间的差异表达基因（DEG, Differentially Expressed Genes）；显著性阈值设定为经Benjamini-Hochberg校正后的错误发现率（FDR）调整p值<0.05。