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| Status |
Public on Nov 09, 2016 |
| Title |
Immunoprecipitated mRNAs with control IgG |
| Sample type |
SRA |
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| Source name |
pancreatic cancer
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| Organism |
Homo sapiens |
| Characteristics |
cell line: S2-013
antibody: control IgG
cell type: pancreatic cancer
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| Extracted molecule |
total RNA |
| Extraction protocol |
Fibronectin-stimulated S2-013 cells were lysed in NP2 buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 0.5% NP-40 and protease and RNAse inhibitors.
RIP protocol: Cell lysates were immunoprecipitated with anti-IGF2BP3 antibody or with normal rabbit IgG and Dynabeads Protein G (Dynal, Oslo, Norway) for 2 h at 4ºC. Beads were pelleted on a magnetic rack (Dynal). The immunoprecipitation products were treated with proteinase K, extracted with phenol-chloroform, and precipitated with ethanol. Purified RNAs were subsequently treated with DNase I (Promega, Madison, WI) and extracted using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Finaly, RNAs were fragmented and purified using an Illumina TruSeq RNA Sample Prep Kit (San Diego, CA) according to the manufacturer’s instructions.
seq protocol: The IGF2BP3-associated mRNAs were identified using a next generation sequencer, Illumina HiSeq 2000 (Hokkaido System Science, Sapporo, Japan).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
library strategy: RIP-Seq
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| Data processing |
The data obtained, Illumina/Solexa reads from the IP (IGF2BP3) experiment and from the control (IgG) were mapped to the mRNAs from RefSeq using the LSKB Database. We approximate the density of each mRNA by the RPKM measure (Reads per kilobase per Million of mapped reads). We normalized RPKM estimates using adjusting the mean and standard deviation of each sample’s log2(RPKM) values.
IlluminaCASAVA ver.1.8.1 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to SeqNovaTMCS (DNANexus).
We approximate the density of each mRNA by the RPKM measure (Reads per kilobase per Million of mapped reads) (Mortazavi A et al, Nat. Methods 5, 621-628, 2008). We normalized RPKM estimates using adjusting the mean and standard deviation of each sample’s log2(RPKM) values.
Supplementary_files_format_and_content: RPKM values for each Sample
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| Submission date |
Jun 03, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Keisuke Taniuchi |
| E-mail(s) |
ktaniuchi@kochi-u.ac.jp
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| Organization name |
Kochi Medical School
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| Street address |
Kohasu
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| City |
Nankoku |
| ZIP/Postal code |
783-8505 |
| Country |
Japan |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE47597 |
RNA immunoprecipitation (RIP) of IGF2BP3 from pancreatic cancer cell extracts cultured on fibronectin |
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| Relations |
| BioSample |
SAMN02189815 |
| SRA |
SRX290630 |
| 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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