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| Status |
Public on Apr 29, 2014 |
| Title |
ACC13_miRNASeq |
| Sample type |
SRA |
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| Source name |
Adrenocortical carcinoma
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| Organism |
Homo sapiens |
| Characteristics |
gender: F
age (years): 45
cell type: Adrenocortical carcinoma
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| Growth protocol |
ES cell–derived NS cells were routinely generated by re-plating d 7 adherent neural differentiation cultures (typically 2–3 × 106 cells into a T75 flask) on uncoated plastic in NS-A medium (Euroclone, Milan, Italy) supplemented with modified N2 and 10 ng/ml of both EGF and FGF-2 (NS expansion medium).
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| Extracted molecule |
total RNA |
| Extraction protocol |
miRNA-Seq libraries are performed from at least 1µg of extracted total RNA with a RIN greater than 7. Before starting, total RNAs are purified with miRNeasy kit wich allows the selection of the small RNA fraction less than 100b. From these samples enriched in small RNAs, libraries are performed according to the protocol published by François Vigneault : Curr Protoc Hum Genet. 2012 Apr;Chapter 11:Unit 11.12.1-10. "High-throughput multiplex sequencing of miRNA"; Vigneault F, Ter-Ovanesyan D, Alon S, Eminaga S, C Christodoulou D, Seidman JG, Eisenberg E, M Church G.
miRNA-Seq libraries are performed from at least 1ug of extracted total RNA with a RIN greater than 7. Before starting, total RNAs are purified with miRNeasy kit wich allows the selection of the small RNA fraction less than 100b. From these samples enriched in small RNAs, libraries are performed according to the protocol published by François Vigneault : Curr Protoc Hum Genet. 2012 Apr;Chapter 11:Unit 11.12.1-10. High-throughput multiplex sequencing of miRNA; Vigneault F, Ter-Ovanesyan D, Alon S, Eminaga S, C Christodoulou D, Seidman JG, Eisenberg E, M Church G. Extraction with Qiacube-miRNeasy 96 Mini (Qiagen) kits; 1 to 10 ug per sample, concentration in range 70-500 ng/uL, volume range 15 to 50 uL, BioAnalyzer profile RIN > 7; Ligation, amplification , quantification and purification according to manufacturer's instructions, with the following details: First, a 3 prime adenylated DNA adaptor is ligated to the enriched sample in the absence of ATP preventing the self-ligation of miRNAs.Then a 5 prime RNA adaptor is ligated in the presence of ATP at the other end of the miRNAs.The RT primer complementary of the 3 prime adaptor is added at this stage with which it will form a duplex thereby reducing the ligation between adaptors. A reverse transcription is therefore perfomed from the RT primer and finally these captured miRNAs are amplified by PCR with primers complementary to the 3 prime and 5 prime adaptors. During this PCR a specific barcode is incorporated allowing individualisation of each library.Each PCR is loaded on the Fragment Analyzer (AATI) for a precise quantification of each miRNA peak of interest.Based on these results a equimolar pool of about ten of different samples are performed. Finally the pooled PCR product is loaded on PAGE in order to excise the band of miRNA that is extracted and purified on a Qiagen MinElute column. Pool Concentration between 0.97 and 4.84 nM per uL; purification quantity is 3500 ng
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
image analysis, base calling and bcl conversion. Evaluation of quality based on Qphred (average score of 20, i.e. e=0.01)
CASAVA demultiplexes multiplexed samples during the bcl conversion step, and converts *bcl files into compressed FASTQ files. It also trim the sequence adaptor
Fastqc software is used to perform quality control checks on raw sequence data
the script "Trim_adapter", provided by mirExpress (http://mirexpress.mbc.nctu.edu.tw/usage.php), handles the sequence files with or without adaptor sequence.
Genome_build: hg19
Supplementary_files_format_and_content: miRanalyzer 0.3 standalone version (http://bioinfo2.ugr.es/miRanalyzer/miRanalyzer.php)
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| Submission date |
Jul 26, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Nabila Elarouci |
| E-mail(s) |
Nabila.Elarouci@ligue-cancer.net
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| Organization name |
Ligue Nationale contre le Cancer
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| Department |
Recherche
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| Lab |
Carte d'identité des tumeurs
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| Street address |
14 rue Corvisart
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| City |
Paris |
| ZIP/Postal code |
75 013 |
| Country |
France |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE49279 |
Micro-RNA sequencing from 78 adrenocortical carcinomas |
| GSE49280 |
Integrated genomic analyses of adrenocortical tumors (SNP array, DNA methylation, mRNA and miRNA expression). |
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| Relations |
| BioSample |
SAMN02265549 |
| SRA |
SRX328518 |
| 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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