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Sample GSM5627656

Query DataSets for GSM5627656

Status

Public on Aug 08, 2023

Title

Sample type

SRA

Source name

Small RNA from extracellular vesicles

Organism

Homo sapiens

Characteristics

culture medium: cultured in DMEM+10%FBS
donor: healthy donor
cell source: bone marrow stromal cells
Sex: M

Growth protocol

Cryopreserved BMSCs at less than passage 3 which were previously isolated from healthy donors using density centrifugation and expanded in Dulbecco’s modified Eagle’s medium (DMEM; Corning) culture medium supplemented with 10% fetal bovine serum (FBS; Gemini Bio-Products) were thawed and expanded again on a CellBIND culture dish (Corning) in the DMEM culture medium. When the cultures attained approximately 80-90% confluency, the cells were harvested by treating with 0.25% trypsin solution for 5 min at 37°C and split into three dishes. After 24 hrs incubation in the DMEM culture medium, the medium in these 3 dishes was replaced with either the DMEM culture medium or the high-performance culture medium (HPM; RoosterNourish-MSC; KT-001, RoosterBio). Once reached 80-90% confluency, the cells were harvested and plated onto 3 dishes at a density of 3,000-4,000 cells/cm2. At 80-90% confluency, the dishes were washed with phosphate-buffered saline (PBS) three times and replaced the media with basal media. After 24 hrs incubation, the conditioned media were collected and centrifuged at 500 g for 5 min to get rid of cellular debris. The supernatant was then filtrated with a 0.22 µm filter (MilliporeSigma). The filtrated conditioned medium was stored in a -80°C freezer.

Extracted molecule

total RNA

Extraction protocol

The filtrated conditioned medium stored at -80°C was thawed and ultracentrifuged at 110,000 g, 4°C for 2.5 hrs to obtain EV pellet using Sorvall Discovery 90SE centrifuge (Hitachi) with AH-629 swinging bucket rotor (ThermoFisher Scientific). The EV pellet was washed with PBS at the same speed for 0.5 hrs. RNA was isolated using mirVana miRNA Isolation Kit (ThermoFisher Scientific) following the manufacturer’s instruction. Specifically, EV pellets isolated from the ultracentrifugation were disrupted with the Lysis Solution. Following organic extraction using acid-phenol: chloroform, total RNA was isolated and eluted in nuclease-free water. RNA quantity and quality isolated from EVs were evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies).
SmRNA-sequencing libraries were prepared from 1.2 to 10 ng of total RNA using the QIAseq miRNA Library Kit (Qiagen, Germantown, MD) as per the manufacturer's instructions. This system offers a built-in Unique Molecular Identifier (UMI) application, which is used to eliminate possible PCR duplicates in sequencing datasets and therefore facilitate unbiased gene expression profiling. The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies). Pooled libraries were diluted to 2 nM in EB buffer (Qiagen) and then denatured using the Illumina protocol.
The denatured libraries were loaded onto an SP flow cell on an Illumina NovaSeq 6000 and run for 71 cycles using a single-read recipe according to the manufacturer's instructions.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina NovaSeq 6000

Description

G674-11

Data processing

De-multiplexed sequencing reads were generated using Illumina bcl2fastq (released version 2.20.0.422, Illumina), allowing no mismatches in the index read. Primary read mapping and UMI analysis were conducted via the GeneGlobe Data Analysis Center (Qiagen).
Genome_build: GRCh38
Supplementary_files_format_and_content: raw read counts are provided in a csv ifle

Submission date

Oct 14, 2021

Last update date

Aug 08, 2023

Contact name

Yuka Imamura Kawasawa

E-mail(s)

yui102@psu.edu

Organization name

Penn State University

Department

College of Medicine, Pharmacology