Transcriptomics of bovine endometrium in oestrus-synchronised beef heifers
During the oestrus cycle, the bovine endometrium undergoes morphological and functional changes, which are regulated by alterations in the levels of oestrogen and progesterone and consequent changes in gene expression. To provide a better understanding of these changes before and subsequent to oestrus, RNA-seq was used to profile the transcriptome of oestrus-synchronized beef heifers. Endometrial samples were collected from 30 animals, which were slaughtered in six groups beginning 12 h after the withdrawal of intravaginal progesterone releasing devices until 7 days after oestrus onset (luteal phase). The groups represented proestrus, early oestrus, metoestrus and early luteal phase.To synchronise oestrus, a controlled internal drug release (CIDR, Pfizer Animal Health) was inserted into all 30 heifers (used in the original study) through the vagina and left for 8 days. The day before CIDR removal the cattle were injected with 2 ml prostaglandins (0.25 mg/ml), (PGF2a-Estrumate, Loughrea, Co., Galway, Ireland) intramuscularly. The cattle were checked for heat behaviour signs every 6 h post-CIDR removal to 60 h post-CIDR removal. All animals came into oestrus within 48–72 h post-CIDR removal (Pluta et al., 2012). blood samples were taken by jugular vein puncture every 12 h on day 0 (the day CIDR was removed), every 6 h on day 1, every 3 h on day 2, every 3 h on day 3, every 12 h on day 4, and once a day from day 5 to day 8. Within 12 h of sampling, serum was separated from plasma and stored at -20°C.Tissue samples were collected from the heifers after they were slaughtered at a local licensed abattoir as the following groups: group 1: 12 h post-CIDR removal (n = 6); group 2: 24 h post-CIDR removal (n = 6); group 3: at the onset of oestrus (n = 4); group 4: 12 h post the onset of oestrus (n = 4); group 5: 48 h post the onset of oestrus (n = 4); and group 6: day 7 after the onset of oestrus (n = 5) (luteal phase). Reproductive tracts were opened longitudinally. Endometrial mucosa was dissected and placed in RNAlater ® (Applied Biosystems, USA), transferred to Eppendorf tubes after 24 h, and frozen at -80°C.Total RNA was isolated from endometrial homogenate using TRIzol reagent (Thermo Fisher) according to the manufacturer's instructions.The RNA integrity number (RIN) was shown to be greater than 7.5 for all samples with an average value of 9.2. The rRNA ratio (28S/18S) varied from 1.1 and 2 between individual samples.RNA-seq library preparation and sequencing were carried out on a commercial basis at Beijing Genomics Institute (BGI), Shenzhen, China (http://www.bgi.com). Briefly, mRNA was isolated from 200 ng total RNA and purified by oligo-dT beads, and the TruSeq RNA Sample Prep Kit v2 (Illumina) protocol was used to construct the libraries.The 'Qualified' libraries were amplified on cBot. The cluster was generated on a flowcell (TruSeq PE Cluster Kit V3–cBot–HS,Illumina). A total of 29 paired-end  2× 90 bp libraries were sequenced with the HiSeq 2000 System (TruSeq SBS KIT-HS V3,Illumina).