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Sample GSM3097993

Query DataSets for GSM3097993

Status

Public on Nov 19, 2018

Title

4m_3_serum

Sample type

SRA

Source name

4 months_serum

Organism

Equus caballus

Characteristics

gestational age: 4 months
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

4m_3_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