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| Status |
Public on May 04, 2012 |
| Title |
Healthy smallRNAseq |
| Sample type |
SRA |
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| Source name |
peripheral blood mononuclear cells
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| Organism |
Homo sapiens |
| Characteristics |
disease: healthy
cell type: peripheral blood mononuclear cells
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| Extracted molecule |
total RNA |
| Extraction protocol |
Peripheral blood mononuclear cells (PBMCs) were isolated with lymphocyte separation medium following the manufacturer’s instruction (Sigama,USA).
Peripheral blood (5 ml) was drawn into EDTA tubes, transferred to the laboratory within 30 min for blood processing.
Total RNA was extracted by Trizol Reagent according to the instructions from the manufacturer (invitrogen, USA).
Small RNA library was prepared according to the prescribed procedure and standards of the Illumina Sample Preparation Protocol.In brief, total RNA was purified by electrophoretic separation on a 15% TBE-urea denaturing PAGE gel and small RNA regions corresponding to the 18-30 nucleotide bands in the marker lane were excised and recovered. The 18-30 nt small RNAs were 5' and 3' RNA adapter-ligated by T4 RNA ligase and at each step length validated and purified by urea PAGE gel electrophoretic separation. The adapter-ligated small RNA was subsequently transcribed into cDNA by SuperScript II Reverse Transcriptase (Invitrogen) and PCR amplified, using primers that anneal to the ends of the adapters. The amplified cDNA constructs, too, were purified and recovered.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
healthy subjects
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| Data processing |
Basecalls performed using CASAVA version 1.4
The 50nt sequence tags from HiSeq sequencing will go through the data cleaning first, which includes getting rid of the low quality tags and several kinds of contaminants from the 50nt tags
Align small RNA tags to the human miRNA precursor/mature miRNA of corresponding species in miRBase17 using BLAST 2.2.25 ,with the following configurations:blastall -p blastn -F F -e 0.01
Normalize the expression of miRNA in two samples to get the expression of transcript per million (TPM). Normalization formula: Normalized expression = Actual miRNA count/Total count of clean reads*1000000
Calculate fold-change and P-value from the normalized expression
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each Sample
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| Submission date |
May 02, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
xiaolin wang |
| E-mail(s) |
wwsdj2002@163.com
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| Organization name |
Zhongshan Hospital, Fudan University
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| Street address |
180 Feng Lin Rd
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| City |
Shanghai |
| ZIP/Postal code |
200032 |
| Country |
China |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE37710 |
MiR-27a-3p in peripheral blood mononuclear cells as a potential marker for pancreatic cancer screening |
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| Relations |
| SRA |
SRX145686 |
| BioSample |
SAMN00990718 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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