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| Status |
Public on Nov 19, 2018 |
| Title |
postpartum_3_chorioallantois |
| Sample type |
SRA |
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| Source name |
postpartum_chorioallantois
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| Organism |
Equus caballus |
| Characteristics |
gestational age: postpartum
tissue: chorioallantois
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| Extracted molecule |
total RNA |
| Extraction protocol |
The RNA derived from chorioallantois was precipitated, then quantified via spectrophotometry (NanoDrop 2000; Thermo Fisher Scientific). Samples with a 260/280 ratio of 1.95 or greater, and a 260/230 ratio of 2.0 or greater underwent further analysis on the Bioanalzyer NanoChip (Agilent Technologies, Santa Clara, CA, USA) to confirm the quality of the RNA. Serum RNA was extracted from serum in a similar manner, with an initial starting ratio of 500 µL of serum to 1 mL of Trizol LS. Quality and purity of the serum RNA was confirmed using the Bioanalyzer PicoChip.
Chorioallantois tissue libraries were created using NEBNext Multiplex Small RNA kit (New England Biolabs, Ipswitch, MA, USA) as per manufacturer’s instructions except where otherwise specified. A total of 250 ng of chorionic RNA was used as input, with all ligations performed for 60 m at 25 ˚C. The cDNA library was amplified using Realtime HiFi master mix (KAPA Biosystems, Wilmington, MA, USA), then amplified for 13 cycles as follows: denatured at 94˚C for 15 s; annealed at 62˚C for 30 s; extended at 70˚C for 15 s, with a final extension at 70 ˚C for 5 m. Samples were then cleaned using AmpureXP magnetic beads (Beckman Coulter Life sciences, Indianapolis, IN, USA), then run on a 6% DNA Retardation gel (LifeTechnologies, Carlsbad, CA, USA). The 150 bp miRNA band was excised, eluted and precipitated in ethanol, then quantified by realtime PCR. Serum RNA libraries were constructed in a similar fashion, although the entire volume of extracted RNA was used as starting material. The NEBNext Multiplex Small RNA kit was used to create the cDNA library as described above, although the 3’ and 5’ adapters were diluted 1:10 prior to ligations. The protocol was otherwise identical except serum libraries were amplified for 20 cycles instead of 13.
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
PP_3_chorioallantois
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| Data processing |
The sequencing results were analyzed using an in-house small RNA analysis program - sRNAanalyzer (http://srnanalyzer.systemsbiology.net). The adaptor sequences were trimmed and low-quality sequences such as low nucleotide complexity reads, homopolymer sequences or di-, tri-nucleotide repeat sequences were removed before the reads were mapped against various databases.
The processed reads were then sequentially mapped against databases including Equus caballus miRNA (mirBase), novel Equus caballus miRNA not yet incorporated in mirBase (horse_novel), transcripts, virus, plant and all miRNA (mirBase), coding transcripts (RefSeq), ribosomal and transfer RNA, equine non-coding RNA, equine coding transcripts (CDS) and equine genomic sequence (DNA)
Genome_build: EquCab 2.0
Supplementary_files_format_and_content: raw_read_counts.xls - excel file
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| Submission date |
Apr 14, 2018 |
| Last update date |
Nov 19, 2018 |
| Contact name |
Shavahn C Loux |
| E-mail(s) |
Shavahn.Loux@uky.edu
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| Organization name |
University of Kentucky
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| Department |
Veterinary Science
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| Street address |
1400 Nicholasville
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| City |
Lexington |
| State/province |
KY |
| ZIP/Postal code |
40546 |
| Country |
USA |
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| Platform ID |
GPL21401 |
| Series (1) |
| GSE113142 |
Normal miRNA Expression Throughout Gestation in the Mare |
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| Relations |
| BioSample |
SAMN08932370 |
| SRA |
SRX3936376 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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