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Sample GSM3061467

Query DataSets for GSM3061467

Status

Public on Sep 30, 2018

Title

Control endometrium mare 2

Sample type

SRA

Source name

endometrium

Organism

Equus caballus

Characteristics

day of pregnancy or estrous cycle: Day 12 of estrous cycle

Treatment protocol

Follicular development and ovulation were monitored routinely by daily transrectal palpation and ultrasound examination. When mares developed an ovarian follicle of approximately 35 mm in diameter, accompanied by prominent endometrial edema, they were treated with 1500 IU human chorionic gonadotropin i.v. (Ovogest®, Intervet Deutschland GmbH, Unterschleissheim, Germany) to induce ovulation. All mares were inseminated artificially with >500 x 10^6 freshly collected, progressively motile, extended spermatozoa from one fertile stallion. Insemination was performed 24 h after induction of ovulation and was repeated if ovulation had not occurred after 48 h. Endometrial samples were obtained by transcervical biopsy. Samples were collected on day 12 after flushing of the uterus. Pregnancy was proved by ultrasonographic detection of an embryonic vesicle in the uterine lumen before flushing. Embryos were flushed transcervically without sedation using up to four times 1.5 l pre-warmed and sterile filtered phosphate buffered saline (DPBS, Lonza Verviers Sprl, Verviers, Belgium). The fluid was recovered directly into sterile glass bottles and subsequently, if necessary, filtered with an embryo filter system. For determination of peripheral plasma progesterone concentrations, blood samples were collected in EDTA tubes from the jugular vein on day 0 and directly after biopsy. In order to analyse tissue composition, the biopsy samples were cut transversely into six equal and plane-parallel slices. For quantitative stereological analyses, every second slice was transferred into embedding capsules. The remaining pieces of the biopsy samples were immediately transferred into vials containing 4 ml RNAlater (Ambion, Huntingdon, Cambridgeshire, UK) for mRNA expression analysis. The vials were cooled on ice and incubated overnight at 4°C. Samples were stored at -80°C until further processing.

Growth protocol

Samples were collected from 4 normal cycling Bavarian Warmblood mares belonging to the Bavarian principal and state stud of Schwaiganger, Germany.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated from the endometrial biopsy samples using Trizol® Reagent (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer’s instructions. Quantity and purity of RNA were measured with a NanoDrop 1000 (PEQLAB Biotechnologie GMBH, Erlangen, Germany). Quality of total RNA was determined electrophoretically with an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). RNA integrity (RIN) values ranged from 8.3 to 9.2.
The mRNA-Seq sample preparation kit (Illumina, San Diego, USA) was used for preparation of RNA-Seq libraries. Library preparation followed the manufacturers instructions. Briefly, poly(A)-containing RNA was purified with oligo-dT-coated magnetic beads starting from 5 µg total RNA and fragmented under elevated temperature using divalent cations. The obtained cleaved RNA fragments were reverse transcribed to first strand cDNA using Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany) and random primers, followed by second strand cDNA synthesis using DNA polymerase I and RNase H. The cDNA fragments underwent an end-repair process with T4 DNA polymerase, Klenow DNA polymerase and T4 Polynucleotide kinase, addition of a single ´A´-base, and ligation of adapters. Ligation products were subsequently separated on a 2% agarose gel (Biozym Phor Agarose, Biozym Scientific GmbH, Hess. Oldendorf, Germany) and a gel slice was cut out (X-Tracta, Biozym Scientific GmbH) in the 200 bp (±25 bp) range. After isolation of the DNA from the gel slice (QIAquick Gel Extraction Kit, Qiagen, Hilden, Germany), cDNA fragments were amplified through 15 cycles of PCR (Phusion DNA polymerase, New England Biolabs GmbH, Frankfurt a. Main, Germany) to generate the final sequencing libraries. Concentration of the cDNA fragments was estimated on an Agilent DNA 1000 chip (Agilent Technologies) and with the Qubit Fluorometer (Invitrogen).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Data processing

Trimmomatic v.0.32.2, remove low quality bases from the 3′ end by sliding window approach (quality score 20 in average across 4 bases), headcrop of 2 bases, remove sequences below a minimal length of 30 nt after the trimming steps
HISAT2 v2.0.3, mapping to the equine genome sequence assembly
QuasR Qcount, generation of read count table for all annotated equine genes based on the NCBI GFF3 annotation file
Counts per million (CPM) per sample filtering tool (Chen et al. 2014), remove sequences with negligible read counts
Genome_build: EquCab2.0
Supplementary_files_format_and_content: tab-delimited text files, Readcounts_whole_endo.txt contains mapped read counts, CPM_Eca_endo_biopTrendedDisp.txt contains counts per million (cpm) values of the genes that passed the CPM filter

Submission date

Mar 22, 2018

Last update date

Sep 30, 2018

Contact name

Stefan Michael Bauersachs

E-mail(s)

stefan.bauersachs@uzh.ch

Organization name

University of Zurich

Department

Department for Farm Animals