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Sample GSM7336643

Query DataSets for GSM7336643

Status

Public on May 14, 2023

Title

143B-test-2

Sample type

SRA

Source name

143B

Organism

Homo sapiens

Characteristics

cell line: 143B
cell type: metastatic osteosarcoma cell line
genotype: IDO OE

Growth protocol

MG63 and 143B cells were cultured in DMEM (Gibco, USA) supplement with 10% (vol/vol) fetal bovine serum (FBS, Gibco, USA), 100 μg/mL penicillin and streptomycin (Gibco, USA) at 37°C. The hFOB1.19 cells were cultured in DMEM/F12 (Gibco, USA) supplement with 10% (vol/vol) FBS (Gibco, USA), 1% (vol/vol) nonessential amino acid solution (Gibco, USA), 0.3 mg/mL G418 and 100 μg/mL penicillin and streptomycin (Gibco, USA) at 33.5°C. Cells were cultured in an atmosphere of 5% CO2 and 90% relative humidity.

Extracted molecule

total RNA

Extraction protocol

Exosomes were isolated from cell culture supernatant with the exoEasy Maxi Kit (Qiagen, No. 76046, USA) according to the manufacturer’s instructions. Total RNA was isolated from exosomes following the protocol of the exoRNeasy SerumKit (Qiagen, No. 77064, USA).
Extracted RNA was used to prepare the miRNA sequencing library with QIAseq miRNA Library Kit (Qiagen, No. 331505, USA).

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina NovaSeq 6000

Data processing

Raw data were generated after sequencing, image analysis, base calling, and quality filtering on the Illumina sequencer. Firstly, Q30 was used to perform quality control. The adaptor sequences were trimmed and the adaptor-trimmed reads (>= 15nt) were left by cutadapt software (v1.9.3). Then, trimmed reads from all samples were pooled, and miRDeep2 software (v2.0.0.5) was used to predict novel miRNAs. The trimmed reads were aligned to the merged pre-miRNA databases (known pre-miRNA from miRBase v22 plus the newly predicted pre-miRNAs) using Novoalign software (v3.02.12) with at most one mismatch. The numbers of mature miRNA mapped tags were defined as the raw expression levels of that miRNA. The read counts were normalized by the edgeR approach.
Supplementary files format and content: The raw and normalized tag counts of the mature miRNA.

Submission date

May 12, 2023

Last update date

May 14, 2023

Contact name

Dan Yang

E-mail(s)

yangdanmuzi@126.com

Organization name

Shanghai Children's Hospital

Street address

355 Luding Road, Putuo District