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| Status |
Public on Jan 19, 2019 |
| Title |
TSMC_3_miRNA-seq |
| Sample type |
SRA |
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| Source name |
Tracheal smooth muscle cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Tracheal smooth muscle cells
cell subtype: Primary isolated cells
strain: C57BL/6J
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| Treatment protocol |
No treatment
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| Growth protocol |
The isolated primary cells were cultured in DMEM/F12 containing 10% fetal bovine serum (FBS) at 37°C with 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA (including small RNA) was extracted using TRNzol (Tiangen, Beijing, China).
RNA libraries were prepared for sequencing using standard BGI protocols
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
BGISEQ-500 |
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| Data processing |
Experiment Pipeline Steps for Small RNA Sequencing: 1) Filter small RNA: separate 18-30nt RNA segment by PAGE gel. 2) 3' adaptor ligation: link 5-adenylated, 3-blocked single-stranded DNA adapter to 3' end of selected small RNAs from step 1. 3) Reverse primer annealing: the RT primer is added to the solution from step 2, and is cross-linked to the 3' adapter of RNAs and excessive free 3' adapter. 4) 5' adaptor ligation: the 5' adaptor is linked to the 5' end of the product from step 3. The connection (5鈥 adaptor) will be attached to the 5鈥 end only, but it is not c onnected to the 3' adaptor or RT primer hybrid chain. This greatly reduces self-ligation. 5) Strand cDNA synthesis: reverse extend the RT primer in step 3 to synthetize strand cDNA. 6) PCR amplification: use high-ping polymerase to amplify cDNA, enrich cDNA with both 3' and 5' adaptor. 7) Library fragment selection: separate the PCR product of 100~120bp by PAGE gel to eliminate primer-dimer and other byproducts. 8) Library quantitative and pooling cyclization.
Bioinformatics Analysis Pipeline for Small RNA Sequencing: 1. Eliminate the low-quality reads, adaptors and other contaminants to get clean reads 2. Summarize the length distribution of the clean tags, common and specific sequences between samples 3. Annotate the clean tags into different categories 4. Predict the novel miRNA 5. Function annotation of known miRNAs
Data Filtering: 1) Remove low quality tags 2) Remove tags with 5' primer contaminants 3) Remove tags without 3' primer 4) Remove tags without insertion 5) Remove tags with poly A 6) Remove tags shorter than 18nt 7) Summarize the length distribution of the clean tags. After filtering, the remaining tags are called 'clean tags' and stored in FASTQ format.
Reads Mapping: Bowtie2 : -q -L 16 --phred64 -p 6 cmsearch: --cpu 6 --noali
sRNA Classification: MiRbase > pirnabank > snoRNA(human/plant) > Rfam > other sRNA. sRNA Prediction: miRDeep2, Piano,
DEGseq: Fold Change >= 2 and Adjusted Pvalue <= 0.001
Genome_build: mm10
Supplementary_files_format_and_content: counts + TPMs
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| Submission date |
Jan 18, 2019 |
| Last update date |
Jan 20, 2019 |
| Contact name |
Su-Ren Chen |
| E-mail(s) |
chensuren@ioz.ac.cn
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| Organization name |
Institute of Zoology, Chinese Academy of Sciences
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| Department |
State Key Laboratory of Stem Cell and Reproductive Biology
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| Street address |
1Beichen West Road, Chaoyang District
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| City |
Beijing |
| ZIP/Postal code |
100101 |
| Country |
China |
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| Platform ID |
GPL23479 |
| Series (2) |
| GSE125302 |
miRNA-Seq of testicular peritubular myoid cells, other testicular cells, and other tissue smooth muscle-like cells |
| GSE125304 |
RNA-seq and miRNA-seq of testicular peritubular myoid cells, other testicular cells, and other tissue smooth muscle-like cells |
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| Relations |
| BioSample |
SAMN10769186 |
| SRA |
SRX5260762 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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