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Sample GSM4240586

Query DataSets for GSM4240586

Status

Public on Jul 01, 2021

Title

ExoC4-miRNA

Sample type

SRA

Source name

plasma exosome

Organism

Homo sapiens

Characteristics

tissue: plasma exosome
gender: female
disease state: healthy control women

Treatment protocol

Exosome was isolated from plasma using ExoquickTM reagent (EXOQ5A1, System Biosciences) according to the manufacturer’s instructions. Briefly, add 1/4 volume of ExoQuick Solution into plasma to precipitate exosomes. After incubating for at least 30 min at 4°C, the mixture was centrifuged at 1500 × g for 30 min, supernatant was discarded and tubes were centrifuged again (1500 g/5 min). The pellet was re-suspended in 100 µL of sterile PBS, and stored at −80°C.

Growth protocol

Blood was sampled on day 3 to day 5 of menstruation. Collect whole blood into commercially available EDTA-treated tubes. Centrifugate for 10 minutes at 2500 x g.The resulting supernatant is plasma.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure
Approximately 1 µg of total RNA were used to prepare nine small RNA librariesaccording to the protocol of TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA, USA). And then the libraries were sequenced by Illumina Hiseq 2500 at the LC-BIO following the vendor’s recommended protocol. Raw reads were subjected to an in-house program, ACGT101-miR (LC Sciences, Houston, Texas, USA) to remove adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, snoRNA) and repeats.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2500

Data processing

Unique sequences with length in 18~25 nucleotide were mapped to specific species precursors in miRBase 22.0 by BLAST search to identify known miRNAs and novel 3p- and 5p- derived miRNAs.
Length variation at both 3' and 5' ends and one mismatch inside of the sequence were allowed in the alignment.
The unique sequences mapping to specific species mature miRNAs in hairpin arms were identified as known miRNAs. The unique sequences mapping to the other arm of known specific species precursor hairpin opposite to the annotated mature miRNA-containing arm were considered to be novel 5p- or 3p-derived miRNA candidates.
The remaining sequences were mapped to other selected species precursors (with the exclusion of specific species) in miRBase 22.0 by BLAST search, and the mapped pre-miRNAs were further BLASTed against the specific species genomes to determine their genomic locations.
The above two we defined as known miRNAs. The unmapped sequences were BLASTed against the genomes, and the hairpin RNA structures containing sequences were predicated from the flank 120 nt sequences using RNAfold software (http://rna.tbi.univie.ac. at/cgi-bin/RNAfold.cgi). The criteria for secondary structure prediction were: (1) number of nucleotides in one bulge in stem (≤12) (2) number of base pairs in the stem region of the predicted hairpin (≥16) (3) cutoff of free energy (kCal/mol ≤-15) (4) length of hairpin (up and down stems + terminal loop ≥50) (5) length of hairpin loop (≤200). (6) number of nucleotides in one bulge in mature region (≤4) (7) number of biased errors in one bulge in mature region (≤2) (8) number of biased bulges in mature region (≤2) (9) number of errors in mature region (≤4) (10) number of base pairs in the mature region of the predicted hairpin (≥12) (11) percent of mature in stem (≥80).
Genome_build: mirbase 22
Supplementary_files_format_and_content: miRNA expression profiles

Submission date

Jan 01, 2020

Last update date

Jul 01, 2021

Contact name

Xiao Jiang

E-mail(s)

jx494329398@gmail.com

Organization name

Center for Reproductive Medicine, Shandong University

Street address

157 Jingliu Road