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Sample GSM5061276

Query DataSets for GSM5061276

Status

Public on Jan 16, 2024

Title

C27

Sample type

SRA

Source name

Extracellular vesicle

Organism

Homo sapiens

Characteristics

tissue: Blood plasma
disease state: Healthy

Treatment protocol

For each blood collection, 2 ml venous blood was collected into 10 ml BD K2EDTA Vacutainer tubes to prevent coagulation. The anticoagulant-treated blood samples were immediately inverted several times and transferred to 2 ml conical tubes (Eppendorf; Cat# 0030120094). To obtain plasma, blood samples were centrifuged at 1,300 × g for 10 min at room temperature (RT). The upper layer containing plasma was transferred to new tubes and centrifuged twice at 2,500 × g for 15 min at RT to obtain platelet poor plasma (PPP). Each plasma supernatant was carefully transferred to 1 ml fresh tubes and then stored at -80 °C until further use.

Extracted molecule

total RNA

Extraction protocol

Frozen plasma samples were thawed in thermostat water bath for 2 min at 37 °C and then centrifuged at 2,500 × g for 15 min at 4 °C to remove precipitated proteins and lipids. ~0.5-1 ml plasma was used as the starting material for each EVs isolation by exoRNeasy Midi kit (Qiagen; Cat#77144) according to the manufacturer’s instructions. For EV-RNA extraction, 1 ml Trizol reagent (Invitrogen; Cat#15596026) was added to the column and centrifuged at 3,000 × g for 1 min. Total EV-RNA was isolated by Trizol reagent according to manufacturer’s recommendations.
A total amount of 0.5-1 ng RNA per sample was used as input material for the RNA sample preparations. The cDNA libraries establishment of small RNAs included adaptor ligation, adapter depletion, first-strand cDNA synthesis, Cas9/sgRNA in vitro cleavage treatment, PCR amplification and gel purification.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina NovaSeq 6000

Data processing

We used cutadapt to clip adaptor and filter low quality reads. Reads failing to match the adaptor or reads with lengths shorter than 17 nt were discarded. Redundant sequences were collapsed as useful reads for further analysis. Then, we aligned the reads to reference sequences by bowtie. To assess the expression levels of the miRNAs, the 3’ ends of miRNA with ≤ 3 nt deletions or additional sequences derived from pri-miRNAs were counted as miRNAs. The miRNA expression level was normalized by size factor from DEseq2.
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text files include log2-transformed normalized read counts for each sample

Submission date

Feb 03, 2021

Last update date

Jan 16, 2024

Contact name

Wei Liu

E-mail(s)

liuwei2018@sibcb.ac.cn

Organization name

Chinese Academy of Sciences

Department

Institute of Biochemistry and Cell Biology