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Sample GSM6172132

Query DataSets for GSM6172132

Status

Public on May 01, 2024

Title

Embryonic Stem cell H9 derived exosome_rep3

Sample type

SRA

Source name

Embryonic stem cell strain H9

Organism

Homo sapiens

Characteristics

cell line: Embryonic stem cell strain H9

Treatment protocol

No specific treatment were done during the sample collection.

Growth protocol

1%FBS low-glucose DMEM were used as culture medium for mesenchymal stem cell and osteoblast cell culture, while embryonic stem cells were cultured using PSGro serum-free medium. 1% FBS were used to generate differentiation of embryonic stem cells. When cells reach 70% confluent, the cells were ready to perform exosome collection. For exosome collection, the medium were collected at a 3-days interval for exosome collection.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer's procedure
Approximately 5 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

Description

small extracellular vesicles

Data processing

Subsequently, 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).
Assembly: Homo_sapiens.GRCh38
Supplementary files format and content: excel,expression profiles

Submission date

May 20, 2022

Last update date

May 01, 2024

Contact name

Chen Xu

E-mail(s)

chenxu8836@hotmail.com

Phone

+86-13774294166

Organization name

The Second Military Medical University