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| Status |
Public on Mar 07, 2013 |
| Title |
Day 14 non-pregnant control, biological replicate 1 |
| Sample type |
SRA |
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| Source name |
endometrial tissue sample
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| Organism |
Sus scrofa |
| Characteristics |
gender: female
Stage: day 14 of estrous cycle
organ/tissue: uterus/endometrium
breed: German Landrace
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| Treatment protocol |
The uteri were removed and each uterine horn was subsequently opened longitudinally at the anti-mesometrial side. Hyperemic zones, the sites of embryonic attachment, were visual in the endometrium on the mesometrial side. In pregnant sows, samples of the endometrium (containing the lamina epithelialis, lamina propria and tela submucosa, but not tunica muscularis) were taken from hyperemic zones (see Supplemental Figure 1 for examples of hyperemic zones). Samples were taken from three locations of each uterine horn, proximal (the end, close to the ovaries), medial, and distal (next to the corpus uteri). Samples from the endometrium of the non-pregnant sows were taken from comparable locations. Tissue samples for isolation of RNA were immediately transferred to RNAlater™ (Ambion, Huntingdon, Cambridgeshire, UK), incubated overnight at 4°C and stored at -80°C until further use.
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| Growth protocol |
Prepuberal German Landrace gilts obtained a single injection of 750 IU PMSG (Intergonan®, MSD Animal Health Innovation GmbH, Schwabenheim, Germany) and 72 h later 500 IU hCG (Ovogest®, MSD Animal Health Innovation GmbH) to synchronize ovulation. Gilts of the “pregnant” group were inseminated twice (24 h and 36 h after hCG) with a standard dose of Pietrain semen whereas gilts of the “non-pregnant” control group were inseminated with the supernatant of centrifuged (10 min, 3,000 rpm) semen of the same boar. Gilts were slaughtered on day 14 after insemination.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from endometrium using TRIzol® (Invitrogen, Carlsbad, CA, USA) according to the manufacturers recommendations. Purity (based on 260nm:280nm and 260nm:230nm ratios) and quantity of the obtained total RNA was measured by use of a NanoDrop ND-1000 (PEQLAB Biotechnologie GmbH, Erlangen, Germany). Integrity of the RNA was assessed by analysis on Agilent RNA Nano 6000 microfluidic chips with an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). RNA integrity numbers (RIN) ranged from 7.0 to 8.8.
Equal amounts of total RNA from samples derived from proximal, medial and distal endometrial sections of one uterine horn were pooled for each animal. 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).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
Pool of RNAs from endometrial tissue samples collected from the distal, medial and proximal part of one uterine horn
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| Data processing |
Basecalls performed using CASAVA version 1.6
Trimming: 3' ends were trimmed using Galaxy FASTQ Quality Trimmer, window size: 5, step size: 1, maximum number of bases to exclude from the window during aggregation: 0, aggregate action for window: min. score, trim until aggregate score is: >=30
Trimmed sequences were filtered according to a minimal length of 30 bp with Galaxy Filter FASTQ
Mapping to genome assembly Sscrofa10.2 with Tophat 1.4, tophat parameters: Input Parameter Value Will you select a reference genome from your history or use a built-in index? indexed Select a reference genome sscrofa_10.2_stefan Is this library mate-paired? single RNA-Seq FASTQ file 224: Filter FASTQ on merged 909_Pr TopHat settings to use full Library Type FR Unstranded Anchor length (at least 3) 15 Maximum number of mismatches that can appear in the anchor region of spliced alignment 0 The minimum intron length 50 The maximum intron length 500000 Allow indel search Yes Max insertion length. 3 Max deletion length. 3 Maximum number of alignments to be allowed 5 Minimum intron length that may be found during split-segment (default) search 50 Maximum intron length that may be found during split-segment (default) search 500000 Number of mismatches allowed in the initial read mapping 1 Number of mismatches allowed in each segment alignment for reads mapped independently 1 Minimum length of read segments 25 Use Own Junctions Yes Use Gene Annotation Model Yes Gene Model Annotations 227: Ssc10_2_EntrezGene.gtf Use Raw Junctions No Only look for supplied junctions No Use Closure Search No Use Coverage Search Yes Minimum intron length that may be found during coverage search 50 Maximum intron length that may be found during coverage search 20000 Use Microexon Search No
Cufflinks 1.3, Max Intron Length 300000 Min Isoform Fraction 0.05 Pre MRNA Fraction 0.05 Perform quartile normalization No Use Reference Annotation Yes Reference Annotation 227: Ssc10_2_EntrezGene.gtf Use Reference Annotation for RABT assembly No Ignore reads come from this annotation file No Perform Multiread correction Yes Perform Bias Correction Yes Reference sequence data cached Set Parameters for Paired-end Reads? (not recommended) No
Cuffmerge for generation of a GTF file for transcript annotation
RPKM calculator; calculates RPKM (reads per kilobase per exon model) and number of reads mapped for each gene/transcript from a BAM file based on a given GTF annotation file. GTF from Cuffmerge was used as annotation file. Read counts were subsequently used for statistical analysis with DESeq.
Genome_build: Sscrofa10.2/susScr3
Supplementary_files_format_and_content: txt files, containing columns for transcript consensus ID, Entrez Gene ID, original transcript ID (Refseq ID or Cufflinks ID), nearest Refseq ID, number of mapped reads
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| Submission date |
Jan 22, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Stefan Michael Bauersachs |
| E-mail(s) |
[email protected]
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| Organization name |
University of Zurich
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| Department |
Department for Farm Animals
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| Lab |
Genetics and Functional Genomics
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| Street address |
Eschikon 27 EHB 23.1
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| City |
Lindau |
| State/province |
Zurich |
| ZIP/Postal code |
8315 |
| Country |
Switzerland |
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| Platform ID |
GPL15064 |
| Series (1) |
| GSE43667 |
Deep Sequencing of the Porcine Endometrial Transcriptome on Day 14 of Pregnancy |
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| Relations |
| SRA |
SRX218947 |
| BioSample |
SAMN01893934 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1067871_D14C_902_read_counts.txt.gz |
320.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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