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| Status |
Public on Feb 14, 2017 |
| Title |
HuR-D226A (asp-aln mutant)_133 |
| Sample type |
SRA |
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| Source name |
HuR-D226A (asp-aln mutant), UM-SCC 74B cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: University of Michigan Squamous Cell Carcinoma cell line 74B (UM-SCC 74B)
transfection: GFP plasmid vector containing HuR-D226A
rna ip: Isotype Control IgG (Santa Cruz, Cat# sc-2025)
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| Treatment protocol |
The GFP plasmid vectors contained full-length HuR, HuR-D226A, HuR-CP1. The plasmid transfections were carried out using Lipofectamine 2000 (Invitrogen).
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| Growth protocol |
The oral cancer cell lines were maintained in DMEM with high glucose, 10% fetal bovine serum, penicillin-streptomycin-L-glutamine in a humidified 5% CO2 environment at 37°C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
HuR RNP IP was performed as previously described. Briefly, cell lysates were prepared from exponentially growing oral cancer cells that had been treated with IR or left untreated as a control. Equal amounts of protein were used (500-1000 μg). HuR monoclonal antibody 3A2 (Santa Cruz), GFP antibody (Clontech), or isotype control IgG (Sigma) were pre-coated onto Protein A/G Sepharose beads (PAS). Lysates were pre-absorbed with IgG (30 μg) and then removed by the addition of PAS beads. Individual pull-downs were performed at 4°C for 1–2 h to minimize the potential re-assortment of mRNAs. For RNA analysis, the beads were incubated with 1 ml of NT2 buffer containing 20 U of RNase-free DNase I (15 min, 30°C), washed twice with 1 ml of NT2 buffer, and further incubated in 1 ml of NT2 buffer containing 0.1% SDS and 0.5 mg/ml proteinase K (15 min, 55°C) to digest the proteins bound to the beads. RNA was extracted using phenol and chloroform and precipitated in the presence of glycogen.RNA was harvested using Trizol reagent. 100 ng of total RNA from RIP samples was used to prepare RNA-Seq libraries using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA), following the protocol described by the manufacturer.
RNA libraries were prepared for sequencing using standard Illumina protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiScanSQ |
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| Description |
133
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| Data processing |
Illumina Casava1.8 software used for basecalling.
Sequenced reads (fastq files) were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence
Secondary analysis was carried out on an OnRamp Bioinformatics Genomics Research Platform (OnRamp Bioinformatics, San Diego, CA). OnRamp’s advanced Genomics Analysis Engine utilized an automated RNAseq workflow to process the data, including data validation and quality control and read alignment to the human genome (hg19) using TopHat2
The resulting SAM files were sorted and inputted into Partek® Genomics Suite® to generate count data for gene-level differential expression analyses using the Partek built-in RNAseq workflow.
One-way Analysis of Variance (ANOVA) was performed on all sample comparisons using the Partek RNAseq pipeline. All factors were treated as fixed factors for control and treated samples. All data were treated as a balanced design where the number of samples in each group (control vs treatment) were equal.
Genome_build: HG19
Supplementary_files_format_and_content: tab-delimited text files include RPKM values, fold change, etc.
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| Submission date |
Mar 22, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Gary Hardiman |
| E-mail(s) |
[email protected]
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| Phone |
8437920771
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| Organization name |
MUSC
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| Street address |
135 Cannon Street, Suite 303 MSC835
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| City |
Charleston |
| State/province |
SOUTH CAROLINA |
| ZIP/Postal code |
29425 |
| Country |
USA |
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| Platform ID |
GPL15456 |
| Series (1) |
| GSE79477 |
Interaction between cyclooxygenase-2 and caspase-3 abolishes the cleavage of RNA-binding protein HuR and promotes drug resistance in oral squamous cell carcinoma |
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| Relations |
| BioSample |
SAMN04572163 |
| SRA |
SRX1652632 |
| 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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