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| Status |
Public on Apr 04, 2014 |
| Title |
10Hvvs |
| Sample type |
SRA |
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| Source name |
day 10 hatched embryo pool
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| Organism |
Bos taurus |
| Characteristics |
day: 10
Stage: hatched embryo
superovulated: Yes
pooled: Yes
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| Treatment protocol |
In order to determine the precise temporal relationship between stages of conceptus development and transcript profiles, the key time points in development examined were Day 7 (blastocyst formation), Day 10 (posthatch- ing spherical conceptus), Day 13 (ovoid conceptus, initiation of elongation), Day 16 (filamentous conceptus, day of maternal recognition) and Day 19 (filamentous conceptus, initiation of implantation). Following slaughter on a given day, the reproductive tract of each heifer was recovered within 30 min of slaughter and placed on ice. Both uterine horns were flushed with 40 ml PBS supplemented with 3% fetal calf serum to recover the embryos. All embryos were washed in PBS and then immediately frozen at -80 degrees C with a minimum volume of PBS until processed for RNA extraction. Only morphologically intact blastocysts/conceptuses were selected and used.
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| Growth protocol |
Synchronization of animals to collect in vivo-derived bovine embryos was carried out as previously described. Briefly, the estrous cycles of crossbred beef heifers (approximately 18 mo old, predominantly Charolais and Limousin cross; n 1⁄4 72) were synchronized using an 8-day Controlled Internal Drug Release (CIDR) device treatment (1.94 g progesterone; InterAg, Hamilton, New Zealand) with administration of a prostaglandin PGF2a analogue (Estrumate; Shering-Plough Animal Health; containing 0.5 mg cloprostenol) the day before CIDR removal to ensure complete regression of the corpus luteum. Animals were checked regularly, and only those heifers that came into standing estrus (Day 0) within a narrow window (36–48 h after CIDR removal) were used for the experiment. Based on our predicted embryo recovery rates at the different stages, heifers were assigned randomly to five experimental groups for embryo recovery on a specific day: group 1 (Day 7; n 1⁄4 9 animals), group 2 (Day 10; n 1⁄4 13), group 3 (Day 13; n 1⁄4 23), group 4 (Day 16; n 1⁄4 16), and group 5 (Day 19; n 1⁄4 15). In order to ensure sufficient numbers of blastocysts to work with on Day 7 and Day 10, the heifers assigned to those days were superovulated. Starting on Day 10 of the estrous cycle, heifers in groups 1 and 2 were superovulated with FSH given as twice-daily injections over 4 days, and luteolysis was induced with a second PG injection on Day 12, while heifers in groups 3, 4, and 5 were not superovulated. All heifers were kept under identical conditions and inseminated with semen from the same bull with proven fertility.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Day 7 and Day 10 blastocysts were pooled into groups of five, while individual conceptuses from Days 13, 16, and 19 were used for mRNA extraction. Polyadenylated mRNA was isolated from Day 7, Day 10, and Day 13 blastocysts/conceptuses by two rounds of selection using the Dynabeads mRNA DIRECT Kit (Invitrogen). However, for Day 16 and Day 19 conceptuses, initially total RNA was isolated using the TRIzol reagent (Invitrogen) per the manufacturer’s instructions. On-column DNase digestion and RNA cleanup was performed using an RNeasy Midi Kit (Qiagen) followed by two rounds of polyadenylated mRNA selection using Dynabeads mRNA DIRECT Kit (Invitrogen). Each developmental stage was represented by five biological replicates that were processed independently.
RNA libraries were prepared for sequencing using standard Illumina protocols
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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 |
embryo.d10.10Hvvs
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| Data processing |
basecalled using Illumina software
aligned to bovine genome using Illumina ELAND software using bt4 genome and ensembl bovine annotation version 54
read counts for each sample were generated using Illumina CASAVA software
Reads per kilobase of exon per million mapped sequence reads (RPKM) was calculated.
Genome_build: Btau_4.0; Ensembl bovine genome annotation version 54
Supplementary_files_format_and_content: tab delimited text files give RPKM counts for each sample
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| Submission date |
Apr 04, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Paul McGettigan |
| E-mail(s) |
pmcget02@gmail.com
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| Organization name |
University College Dublin
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| Department |
School of Agriculture, Food Science and Veterinary Medicine
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| Lab |
Animal Genomics Laboratory
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| Street address |
Veterinary Sciences Centre, UCD
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| City |
Belfield |
| State/province |
Co Dublin |
| ZIP/Postal code |
Dublin 4 |
| Country |
Ireland |
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| Platform ID |
GPL15750 |
| Series (1) |
| GSE56513 |
RNA sequencing reveals novel gene clusters in bovine conceptuses associated with maternal recognition of pregnancy and implantation |
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| Relations |
| BioSample |
SAMN02719422 |
| SRA |
SRX511053 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1363037_embryo.d10.10Hvvs.rpkm.txt.gz |
245.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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