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Sample GSM3109214 Query DataSets for GSM3109214
Status Public on Feb 28, 2019
Title MSCATV-Exo-1: exosomes derived from ATV pretreated MSCs
Sample type SRA
Source name bone-marrow derived MSCs
Organism Mus musculus
Characteristics strain: SD
gender: male
tissue: exosomes derived from ATV pretreated MSCs
cell type: Mesenchymal stem cells (MSCs)
treatment: ATV (1μM)
time: 24 hours
sample type: 24 hours
Treatment protocol For isolation of exosomes derived from MSCs, 10 15cm-dishes of MSCs were pretreated with 1 μmol/L ATV or same volume of DMSO in exosome-free IMDM for 48 h until collection of the conditioned medium. Cells and debris were removed by differential centrifugation at 300 g for 10 min, 2,000 g for 20 min respectively; and macrovesicles were removed at 13,500 g for 30 min. The supernatant continue to be ultracentrifuged at 120,000 g for 70 minutes to obtain the crude exosome pellet. The pellet was subsequently washed with phosphate buffered saline (PBS pH7.4) followed by repeat ultracentrifugation for 70 minutes at the same speed.
Growth protocol Rat MSCs were isolated from bone marrow of 6–8 week old male SD rats. Rats were sacrificed by isoflurane inhalation and cervical dislocation. In brief, total bone marrow cells were flushed with culture medium from the femur and tibia and seeded in culture dishes containing Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 U/mL)/streptomycin (100 mg/mL). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. The first medium change was performed to remove the non-adherent cells at 24 hours. Fresh complete medium was added and replaced every 3 days. Each primary culture was sub-cultured 1:3 when MSCs grew to approximately 80% - 90% confluence. MSCs at Passages 3-4 were used.
Extracted molecule total RNA
Extraction protocol A total of 200 ml of conditioned media of each sample was mixed with Ribo™ Exosome Isolation Reagent and exosomes isolation was performed according to the manufacturer's instructions (RiboBio, Guangzhou, China).Total RNA was quantified by the NanoDrop ND-2000 (Thermo Scientific)and the RNA integrity was assessed using Agilent Bioanalyzer 2100 (Agilent Technologies).Total RNA from exosomes was used for library preparation and sequencing.
Library preparation and sequencing were performed at RiboBio.Briefly, RNA was fragmented to approximately 200 bp. Subsequently,the collected RNAs were subjected to first strand and second strand cDNA synthesis followed by adaptor ligation and enrichment with a low-cycle according to the instructions provided with the NEBNext® Ultra™ RNA Library Prep Kit for Illumina (NEB, USA). The purified library products were evaluated using the Agilent 2200 TapeStation (Agilent Technologies) and Qubit®2.0 (Life Technologies, USA)and then sequencing using a HiSeq30000.
Library strategy ncRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina HiSeq 3000
Data processing Illumina RNA-seq was performed by the RiboBio Company (Guangzhou, China).
Quality assessment of the RNA-seq data was done using FASTX-Toolkit. Reads with >20% nucleotides with Phred quality scores <20 were removed from further analysis.
Quality-filtered reads were then aligned to the rat reference genome RGSC 6.0/rn6 using the TopHat2 aligner using default settings. Reads mapped to ribosomal RNA were removed by TopHat2. Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Mortazavi A, et al., Nat Methods. 2008.
GFOLD was used for ranking differentially expressed genes from RNA-seq data. Differential lncRNA expression analysis was performed using the method described by Audic S and Claverie JM., Genome Res. 1997. False discovery rate (FDR) (Benjamini Y and Hochberg Y., Journal of the Royal Statistical Society. 1995) was used to determine the p-value thresholds in multiple testing . Cutoff values of fold change >1.5 and Q value <0.001 were then used to select for differentially expressed genes between sample group comparisons.
Genome_build: Rnor_6.0
Supplementary_files_format_and_content: tab-delimited text files include RPKM values, gene type, chromosome, and strand information for each Sample.
Submission date Apr 24, 2018
Last update date Feb 28, 2019
Contact name Peisen Huang
Phone 4043844891
Organization name Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College
Street address No.167 Bei Li Shi Road, Xicheng District
City Beijing
State/province Beijing
ZIP/Postal code 100037
Country China
Platform ID GPL21493
Series (1)
GSE113570 Exosomal lncRNA sequencing in Atorvastatin treated and non-treated MSC-derived exosomes
BioSample SAMN08975020
SRA SRX3989918

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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