 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 18, 2021 |
| Title |
small_RNA_IVF.Mo.1 |
| Sample type |
SRA |
| |
|
| Source name |
IVF morula stage embryos
|
| Organism |
Bos taurus |
| Characteristics |
developmental stage: Morula stage
production method: In vitro fertilization
molecule subtype: RNA < 200 nt in length
|
| Growth protocol |
Following 22 to 24 hr of maturation, MII oocytes were fertilized using the laboratory’s standard in vitro fertilization protocol [46]. Briefly, one straw of cryopreserved bovine semen obtained from a Holstein bull (Hoffman AI, Logan, Utah) was removed from the liquid nitrogen tank and placed into a 35C water bath to thaw. Live sperm were isolated by centrifugation through a 45%/90% percoll gradient (MP Biomedical, Irvine, California), suspended (final concentration 1x106/ml) in Tyrode’s albumin lactate pyruvate containing 10 microgram/ml procine heparin and used to fertilize the oocytes. Twenty to 22 hr post-IVF, cumulus cells were removed by vortexing, and fertilized zygotes were washed in phosphate buffered saline with 0.32 mM sodium pyruvate, 5.55 mM glucose, and 3 mg/ml bovine serum albumin (PB1+). After washing, zygotes were cultured on a monolayer of bovine cumulus cells in 50 microliters of synthetic oviductal fluid (SOFaa) [47] with 3% FBS (Hyclone, lot number AC10240545) overlaid with mineral oil. The embryos were cultured at 38.5 deg C in a humidified atmosphere with 5% CO2. Half of the SOFaa medium was removed and replaced with fresh, equilibrated medium every other day starting the day after in vitro culture.
Provided above
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated based on size using the RNA Clean & ConcentratorTM 5 kit (Zymo) from three pools each of oocytes, 2-cell stage embryos, 8-cell stage embryos, morula stage embryos, or blastocyst stage embryos according to the manufacturer’s protocol for purification of small RNA (<200 nt) and large RNA (>200 nt) as separate fractions.
Small RNA sequencing was performed on the Ion Proton Sequencer by using the Ion Total RNA-seq kit v2 (Thermo Fisher Scientific) according to manufacturer’s procedure for small RNA library preparation with no deviations from the specified protocol. By using an RNA isolation protocol that yielded a specific fraction of small RNA, an enrichment step was not needed. Sample volumes were reduced to 3 l by vacuum centrifugation, and the entire sample was used to prepare the small RNA library for sequencing. The cDNA sample was then processed on the Agilent Bioanalyzer (Agilent, Santa Clara, California) to ensure the presence of small RNA bound to a barcode (86 to 106 nt).
|
| |
|
| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Ion Torrent PGM |
| |
|
| Data processing |
Sequence data were processed to remove low quality reads and any artificial reads introduced during library preparation (Perl script Trim Galore, Python script SortMeRNA).
Annotation and abundance analyses of sncRNAs was performed as described previously (Russell, et al. 2017 Reproduction 153: 305-318). Briefly, data processing and bioinformatics analysis for other sncRNA were performed primarily with command line tools available at http://www.smallrnagroup.uni-mainz.de/software.html. First, data were filtered for sequence length between 17 and 32 nucleotides (nt), which includes the canonical size range for mature small RNAs (miRNA, tRFs, piRNA). A separate analysis was performed for sequences between 33-93 nt, which encompassed longer reads characteristic of snoRNA, tRNA and mitochondrial rRNA. This split analysis approach avoided possible skewing of the data when comparing between these diverse sncRNA classes.
Sequence annotation was performed with Unitas v1.6.1, which uses the latest available public small RNA databases to annotate input sequences. Non-annotated sequences were then mapped to the bovine genome (BosTau8) with sRNAmapper to determine which non-annotated sequences were likely of biological, not technical, origin. The mapped sequence reads were redistributed across the genome based on number of mapping locations by using reallocate with the parameters ‘5000 1000 b 0’ (reallocate.pl).
Reallocated map files were then analyzed with ProTRAC v2.4.2, which identifies probable piRNAs through their genomic clustering based on size, sequence, and cluster characteristics (all options at default settings, including repeatmasker and geneset references). Predicted piRNA clusters were compared among samples by using the Galaxy merge tool (usegalaxy.org) to first generate lists of predicted clusters by sample type followed by the join tool to identify overlapping genomic intervals among samples. Sequence logos representing nucleotide biases were generated with ggseqlogo.
Count tables of sncRNAs were generated from the output of Unitas. Normalization and differential expression analysis was performed with the DESeq2 R package. Potential outlier sample replicates were identified by using principal components analyses of the complete sncRNA profile prior to statistical analyses.
Genome_build: UMD3.1
Supplementary_files_format_and_content: txt file for normalized reads for annotated small non-coding RNAs organized by sample ID across columns and annotation in the first column
|
| |
|
| Submission date |
Jun 17, 2021 |
| Last update date |
Jun 18, 2021 |
| Contact name |
Abby D Benninghoff |
| E-mail(s) |
abby.benninghoff@usu.edu
|
| Organization name |
Utah State University
|
| Department |
Animal, Dairy and Veterinary Sciences
|
| Street address |
4815 Old Main Hill
|
| City |
Logan |
| State/province |
UT |
| ZIP/Postal code |
84322 |
| Country |
USA |
| |
|
| Platform ID |
GPL30292 |
| Series (2) |
| GSE178437 |
Dynamics of small non-coding RNAs in bovine scNT embryos through the maternal-to-embryonic transition [non-coding RNA] |
| GSE178438 |
Comparing mRNA and sncRNA profiles during the maternal-to-embryonic transition in bovine IVF and scNT embryos |
|
| Relations |
| BioSample |
SAMN19765347 |
| SRA |
SRX11174703 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
