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Sample GSM6597204

Query DataSets for GSM6597204

Status

Public on Jan 01, 2024

Title

CHMm_rep1

Sample type

SRA

Source name

CHMm

Organism

Canis lupus familiaris

Characteristics

cell line: CHMm

Treatment protocol

At 70~80% confluency in 100mm culture dishes, cell were washed with PBS two times and RPMI media without FBS and antibiotics was replaced.

Growth protocol

Canine mammary gland adenocarcinoma cell lines (CHMp and CHMm) was grown in RPMI 1640 medium (Hyclone) containing 10% fetal bovine serum (FBS, Gibco) and gentamicin (50ug/ml).

Extracted molecule

total RNA

Extraction protocol

Exosomes were purified from cells cultured under serum-free conditions using a combined method of ultrafiltration and ultracentrifuge. First, the culture supernatant was subjected to a differential centrifuge to eliminate cells, dead cells, and cell debris and sequential filtered with 0.45 and 0.22 μm. The filtered supernatant was concentrated using 10K Amicon-Ultra 15 Centrifugal Filter Units (Merck). Filtered units were ultracentrifuged for 80 min and washed with PBS. Exosomes for RNA isolation were lysed with Qiazol (Qiagen) and miRNeasy mini plus kit (Qiagen). Isolated small RNA fracation was directly performed small RNA sequencing.
Small RNA library was prepared using NEXTflex™ Small RNA-Seq Kit v3 following manufacturer's instructions.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 3000

Data processing

To remove adaptors with low-quality reads and extract miRNA-specific sequence, cutadapt was used with options (--quality-base 33 -u 4 -m 22 -M 30 -f fastq -q 20 -O 6 -j 23 -a adapter-sequence
Before and after the trimming step, quality of sequenced reads was estimated using FastQC.
For known and novel miRNA analysis, miRDeep2 package was used. First, all filtered reads data were merged into one file for novel miRNA analysis. The merged data were converted to collapsed fasta-formatted file and aligned to reference genome using mapper.pl script with options (-e -h -j -m -p). Second, novel miRNAs were identified using miRDeep2.pl script. The identified mature and hairpin forms of novel miRNAs were extracted and combined with known forms of miRBase prepared previously. Finally, the expression values of known and novel miRNAs were calculated using the quantifier.pl script. The CPM (counts per million), which is counts scaled by total number of reads, was used for further analysis. To estimate data reproducibility between replicate samples, Pearson correlation values were calculated and visualized using correlation function in R.
Assembly: canFam3.1
Supplementary files format and content: text

Submission date

Sep 22, 2022

Last update date

Jan 01, 2024

Contact name

Je-Yoel Cho

E-mail(s)

jeycho@snu.ac.kr

Organization name

Seoul National University

Department

Biochemistry