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Sample GSM3097998 Query DataSets for GSM3097998
Status Public on Nov 19, 2018
Title postpartum_2_serum
Sample type SRA
 
Source name postpartum_serum
Organism Equus caballus
Characteristics gestational age: postpartum
tissue: serum
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.  
 
Library strategy miRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina NextSeq 500
 
Description PP_2_serum
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
 
Submission date Apr 14, 2018
Last update date Nov 19, 2018
Contact name Shavahn C Loux
E-mail(s) Shavahn.Loux@uky.edu
Organization name University of Kentucky
Department Veterinary Science
Street address 1400 Nicholasville
City Lexington
State/province KY
ZIP/Postal code 40546
Country USA
 
Platform ID GPL21401
Series (1)
GSE113142 Normal miRNA Expression Throughout Gestation in the Mare
Relations
BioSample SAMN08932362
SRA SRX3936384

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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