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Status |
Public on May 01, 2013 |
Title |
3628Ago |
Sample type |
SRA |
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Source name |
BJAB cells_BARTs_RISC-IP
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Organism |
Homo sapiens |
Characteristics |
cell line: BJAB cell type: EBV-negative Burkitt's lymphoma cells ectopic barts: yes (expressing EBV's BART miRNAs) ip antibody: anti-human Ago2 (11A9) antibody ip antibody info: PMID:18430891
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Treatment protocol |
Cell lines grown under the same conditions
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Growth protocol |
BJAB cells were grown in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (R10F) and 200 U/mL penicillin and 200 µg/mL streptomycin at 37°C in a 5% CO2 humidified atmosphere.
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Extracted molecule |
total RNA |
Extraction protocol |
1×10^8 cells were washed with PBS and lysed with 500 µl of PLB buffer (10 mM HEPES, pH 7.0, 100 mM KCl, 0.5% NP-40, 5 mM MgCl2, 200 U/ml RNase inhibitor (Ambion), 1 mM DTT, proteinase inhibitor cocktail) at 4°C for 30 min. Following centrifugation, the lysate was incubated with anti-human Ago2 (11A9) antibody (Provided by Dr. Gunter Meister [Rüdel S, et al. (2008) A multifunctional human Argonaute2-specific monoclonal antibody. RNA 6: 1244–2153.]) conjugated dynabeads (Invitrogen) in 500 µl of NET-2 buffer (20 mM EDTA, pH 8.0, 1 mM DTT, and 200 U/ml RNase inhibitor in NT-2 buffer (150 mM Tris-HCl, pH 7.0, 100 mM Tris-HCl, pH 8.0, 750 mM NaCl, 0.25% NP-40, 5 mM MgCl2)) at 4°C for overnight. The protein-dynabead complexes were washed 6 times with NT-2 buffer, once with NTmS buffer (150 mM Tris-HCl, pH 7.0, 100 mM Tris-HCl, pH 8.0, 0.25% NP-40, 5 mM MgCl2) and resuspended with 1× Turbo DNase buffer and 2 U of Turbo DNase (Ambion) at 37°C for 30 min. The activity of DNase was eliminated with 15 mM of EDTA followed by addition of same volume of proteinase K buffer (2.4 mg/ml of proteinase K and 2% SDS in NT-2 buffer) at 55°C for 30 min. The RNA was extracted with acid-phenol/chloroform (pH 4.5) once, chloroform once and precipitated and resuspended in RNAse-free water. Libraries were generated using Illumina’s mRNA Seq kit (lot#5701543) Rev. D according to manufacturer's protocol. Briefly, samples were fragmented then precipitated at -80°C, followed by first strand & second strand cDNA synthesis. After end repair and adenylation of 3' ends, adapters were ligated to cDNA molecules. Following ligation, cDNA was purified using E-gel Size Select 2% agarose gels. Enrichment of purified cDNA was done via LMPCR (15 cycles) followed by purification using ZymoResearch DNA Clean & Concentrator columns (25ul elution volume). Libraries were validated on Agilent 1000 DNA chip and Quant-iT quantification.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Description |
total RNA after RISC-IP
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Data processing |
Primary analysis performed using RTA 1.12.4.2 followed by secondary analysis with CASAVA 1.8.1 Sequence analysis was conducted using CLC Genomics Workbench Version 4.9; reads were mapped to hg18 human genome build Unique exon reads for each gene were then used to create expression values for each gene with the following formula: Expression Value = (unique exon reads)(10^9)/(exon length)(total unique exon reads) A table was constructed consisting of genes with the respective associated expression values for each condition. Genome_build: hg18 Supplementary_files_format_and_content: text file contains genes and associated enrichment values for each sample. Fold change (Column 4) is the ratio of the respective values BJAB_3628/BJAB_3626 (Column_3/Column_2)
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Submission date |
Apr 30, 2013 |
Last update date |
May 15, 2019 |
Contact name |
Bill Sugden |
E-mail(s) |
sugden@oncology.wisc.edu
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Organization name |
University of Wisconsin-Madison
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Street address |
1111 Highland Ave
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City |
Madison |
State/province |
WI |
ZIP/Postal code |
53705 |
Country |
USA |
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Platform ID |
GPL11154 |
Series (1) |
GSE46514 |
Comparing proteomics and RISC immunoprecipitations to identify targets of Epstein-Barr viral miRNAs [RISC-IP-seq] |
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Relations |
BioSample |
SAMN02087571 |
SRA |
SRX272894 |
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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