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| Status |
Public on May 05, 2012 |
| Title |
CG5099_RIP-Seq, rep2 |
| Sample type |
SRA |
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| Source name |
Drosophila tissue culture cells, CG5099
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2R+
orf encoding protein: CG5099
rip antibody: HA Immunoaffinity Resin (Sigma)
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| Treatment protocol |
Open reading frames for the indicated proteins were inserted into pMK33-C-FLAG-HA acceptor vector. Each clone was transiently transfected into a culture of Drosophila S2R+ cells. Protein expression was induced with 0.35 mM CuSO4.
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| Growth protocol |
Cells were cultured in Schneider's medium at 25˚C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Whole-cell lysates were prepared in lysis buffer (25 mM NaF, 1 mM Na3VO4, 50 mM Tris [pH 7.5], 1.5 mM MgCl2, 125 mM NaCl, 0.2% IGEPAL, 5% glycerol, Complete, and RNase Inhibitor). Each clarified lysate was bound overnight to 75 ml of crosslinked HA immunoaffinity resin (Sigma). Unbound proteins were washed off with lysis buffer followed by PBS, and then bound protein complexes were competitively eluted using synthetic HA peptide YPYDVPDYA (250 mg/ml) in PBS. RNA was extracted and a sequencing library prepared using the Illumina Tru-Seq mRNA Library preparation kits.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
RNA immunoprecipitation (RIP).
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| Data processing |
Library strategy: RIP-Seq
Base calling and demultiplexing was performed using CASAVA 1.8.
Alignments were performed using TopHat version 1.4.0 with the following parameters: -p 8 -G ginormous.100806.gtf --no-novel-juncs -a 6 -m 2 --min-intron-length 28 -I 200000 -F 0 -g 1 --library-type fr-unstranded --transcriptome-index ginormous_transcriptome -x 60 -n 2. Ginormous refers to the annotation published in Graveley, B.R., Brooks, A.N., Carlson, J.W., Duff, M.O., Landolin, J.M., Yang, L., Artieri, C.G., van Baren, M.J., Boley, N., Booth, B.W., et al. (2011). The developmental transcriptome of Drosophila melanogaster. Nature 471, 473–479 (PMID 21179090).
bedGraph files were generated from bam files using bedTools with the following parameters: genomeCoverageBed -bg -split
Genome_build: Dm3
Supplementary_files_format_and_content: Genome browser files are presented in bedGraph format.
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| Submission date |
May 03, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Brenton R Graveley |
| E-mail(s) |
[email protected]
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| Phone |
860-679-2090
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| Organization name |
University of Connecticut Health Center
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| Department |
Genetics and Genome Sciences
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| Street address |
400 Farmington Avenue
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| City |
Farmington |
| State/province |
CT |
| ZIP/Postal code |
06030-3301 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE37756 |
Identification of protein-RNA interactions in Drosophila using RIP-Seq |
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| Relations |
| SRA |
SRX145957 |
| BioSample |
SAMN00990911 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM927205_Obar_BRG_BLUE_FH5443_CG5099_msi_TTAGGC_L006_R1_001_scaled.bedGraph.gz |
26.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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