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Sample GSM6008378

Query DataSets for GSM6008378

Status

Public on Apr 06, 2022

Title

S12 at R+3

Sample type

SRA

Source name

peripheral blood sEVs

Organism

Homo sapiens

Characteristics

tissue: peripheral blood sEVs
time point: R+3
Sex: NA

Extracted molecule

total RNA

Extraction protocol

sEVs were isolated from peripheral blood (PB) samples of using the ExoQuick Plasma preparation and sEV precipitation kit (Cat # EXOQ5TM, System Biosciences, CA, USA). Samples were subsequently centrifuged at 10,000 rpm for 5 minutes, incubated with the exosome precipitation solution and refrigerated at 4ºC for 30 minutes. After centrifugation at 1,500 x g for 30 minutes at 4ºC, a beige-colored pellet was dissolved in sterile 1xPBS.
Total RNA from sEVs was isolated using miRNeasy kit following manufacturer's protocol (Cat#217084, Qiagen). RNA sample quality was assessed by NanoDrop and Agilent 2100 BioAnalyzer. Samples with RNA integrity number (RIN) above 7, OD260/280: 2, and OD260/230 ≥ 2 were used. In addition, RNA degradation and contamination were monitored on 1% agarose gels before RNA-Seq. RNA quality was assessed using an Agilent TapeStation (Agilent, Palo Alto, CA, USA), and RNA concentration was quantified by Qubit 4.0 spectrophotometer. The library for small RNA sequencing was prepared using the Smarter smRNA-seq kit for Illumina (Takara Bio Inc., USA). The quantity and quality of amplified libraries were evaluated using Qubit (Invitrogen, Carlsbad, CA, USA) and Agilent TapeStation high sensitivity D1000 Screen Tape. Small RNA-seq libraries were sequenced using single-end 75 base pairs (SE75) sequencing chemistry on NextSeq 500 instruments following the manufacturer's protocols (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

S12_R+3

Data processing

Illumina bcl2fastq2 Conversion Software v2.20 used for demultiplexing and basecalling.
The 3′ adaptor sequences were removed, and reads with the trimmed length <18 bp were discarded using Cutadapt v2.7. Read quality control was performed with FastQC v0.11.9 http://www.bioinformatics.babraham.ac.uk/projects/fastqc/.
Trimmed and filtered reads were mapped with bowtie (v1.1.1) to the reference genome (GRCh38.p12)
RUVSeq (Risso et al., 2014) was used to correct for batch effects and other unwanted variations. Differentially expressed genes were then identified with edgeR (Robinson et al., 2010). Genes with false discovery rate adjusted p-value (FDR) less than 0.05 and log2 fold change greater than 1 were considered significantly differentially expressed genes.
Assembly: GRCh38.p12
Supplementary files format and content: comma-separated matrix table with CPM counts for every gene and every sample produced with RUVSeq

Submission date

Apr 03, 2022

Last update date

Apr 08, 2022

Contact name

Arsen Arakelyan

E-mail(s)

aarakelyan@sci.am

Organization name

Institute of Molecular Biology NAS RA

Department

Bioinformatics