 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 30, 2018 |
| Title |
Pregnant stroma mare U |
| Sample type |
SRA |
| |
|
| Source name |
endometrium
|
| Organism |
Equus caballus |
| Characteristics |
day of pregnancy or estrous cycle: Day 12 of pregnancy
endometrial cell type/compartment: stroma
|
| Treatment protocol |
The mares were monitored for signs of estrus using a LOGIQ e ultrasound equipment (GE Healthcare, Glattbrugg, Switzerland). When a follicle of at least 35 mm diameter was detected in conjunction with a uterine edema, a single dose of 1500 IU human choriogonadotropin (hCG) was applied to induce ovulation (day -2). The mares were randomly assigned to one of the experimental cycles: control cycle or pregnancy. If assigned to the pregnancy cycle the mare was inseminated with fresh cooled semen one day before ovulation (day -1). To induce luteolysis at the end of the experiment, prostaglandin F2 alpha (PGF2a) was applied on day 12. During the experimental days (-2 to 12) ultrasound images of both uterus and ovaries were recorded and blood samples for measurement of progesterone were collected. On day 12, endometrial samples were obtained by performing a uterine biopsy. After a longitudinal cut of the biopsy, one half was fixated in formalin for histological examination and the other half was snap-frozen in a cryo-embedding matrix (OCT = optimal cutting temperature) in liquid nitrogen and stored at -80°C.
|
| Growth protocol |
Endometrium biopsies for the isolation of specific endometrial cell types were collected from 6 healthy cycling warmblood mares belonging to the University of Zurich, Clinique for Animal Reproduction Medicine, Switzerland. All the experiments with animals were conducted with the permission of the veterinary inspection office of the Kanton Zurich (permission no. 24/2014) and the degree of severity corresponded to grade 1.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Laser capture microdissection (LCM) Stained frozen sections (10 µm) were prepared using a microtome cryostat CryoStar NX50 (Histocom AG, Zug, Switzerland) on PEN-Membrane Glass Slides®, and Arcturus® HistoGene® Frozen Section Staining Kit (Applied Biosystems). The collection of luminal epithelium (LE), glandular epithelium (GE) and stromal areas (S) was performed using an ArcturusXT® Laser Capture Microdissection instrument. RNA isolation, quantification and quality control RNA isolation was performed with the Arcturus PicoPureTM RNA Isolation Kit (Thermo Fisher Scientific, Darmstadt, Germany). Concentration of the RNA was measured with a QuantusTM Fluorometer (Promega; sensitivity 390 pg RNA/ml using QuantiFluor® RNA Dye). Quality was analyzed by the use of the Agilent 2100 Bioanalyzer (Eukaryote Total RNA Pico Assay), which estimates the concentration of the RNA sample and provides a quality score (RNA integrity number, RIN).
For the generation of RNA-Seq libraries from LCM samples, the Ovation® Single Cell RNA-Seq System (NuGEN Technologies, Inc., European office, Leek, The Netherlands) was used starting from 1 ng total RNA. Since for a number of libraries the concentration of the obtained cDNA fragments was too low (Agilent Bioanalyzer DNA High Sensitivity assay), an additional amplification was performed with the KAPA HiFi Library Amplification Kit (Axon Lab AG, Baden, Switzerland). Single-end reads (125 bp) were generated on an Illumina HiSeq 2500 instrument running multiplexed libraries on two lanes of one flow cell.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Trimmomatic v.0.32.2, remove low quality bases from the 3′ end by sliding window approach (quality score 28 in average across 4 bases), remove sequences below a minimal length of 50 nt after the trimming steps
FastqMcf sequence quality filtering and clipping (Galaxy Version 1.0); removal of remaining adapter sequences and sequences below a minimal length of 50 nt after adapter clip
FastUniq v1.1, removal of PCR duplicates
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.txt contains mapped read counts, CPM_Eca_endo_all_dedup_TrendedDisp.txt contains counts per million (cpm) values of the genes that passed the CPM filter, CPM_Eca_endo_sel_TrendedDisp.txt contains counts per million (cpm) values of the genes that passed the CPM filter for selected samples
|
| |
|
| Submission date |
Mar 22, 2018 |
| Last update date |
Sep 30, 2018 |
| Contact name |
Stefan Michael Bauersachs |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Zurich
|
| Department |
Department for Farm Animals
|
| Lab |
Genetics and Functional Genomics
|
| Street address |
Eschikon 27 EHB 23.1
|
| City |
Lindau |
| State/province |
Zurich |
| ZIP/Postal code |
8315 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL21941 |
| Series (1) |
| GSE112236 |
Cell type-specific endometrial transcriptome changes in comparison of equine endometrium samples collected on Day 12 of pregnancy and Day 12 of the estrous cycle |
|
| Relations |
| BioSample |
SAMN08774754 |
| SRA |
SRX3834002 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
