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| Status |
Public on Jun 19, 2017 |
| Title |
Nonspecific Control |
| Sample type |
SRA |
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| Source name |
293HEK cells
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| Organism |
Homo sapiens |
| Characteristics |
virus strain: SINV Toto1101
antibody: Preimmune IgG
rna treatment: RNAse T1
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| Treatment protocol |
Per library, a total of 2E7 293HEK tissue culture cells were infected with SINV Toto1101 at an MOI of 5 PFU/cell. Infection was allowed to proceed for 18 hours prior to UV crosslinking via 5700x100 ujoules per cm2. Purified viral particles, ~1E11 particles in total, were treated similarly.
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| Growth protocol |
293HEK cells were maintained in MEM supplemented with 10% FBS at 37 degrees Celsius in the presence of 5% CO2. Purified SINV particles were obtained from 293HEK cells and purified using a 27% sucrose cushion via ultracentrifugation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Lysates were generated via the addition of RIPA buffer (50mM Tris [pH 7.6] / 150mM NaCl / 1.0% NP-40 / 0.5% Sodium Deoxycholate / 0.1% SDS). The lysates were clarified via centrifugation and immunoprecipitated with either nonspecific control sera, or anti-SINV Capsid polyclonal sera and Protein A agarose beads. Purified RNA:Protein complexes were treated with RNAse T1 for 15 minutes at room temperature, washed several times to remove contaminating sequences and then released from the resin via proteinase K digestion at 37 degrees Celsius for 30 minutes.
The libraries were generated using the NEBNext Small RNA Library Prep Set for Illumina, with the following Index / Sample pairs- (Input, Illumina 13 AGTCAA) (Nonspecific, Illumina 19 GTGAAA) (Cyto Capsid Set A, Illumina 20 GTGGCC) (Cyto Capsid Set B, Illumina 18 GTCCGC) (Capsid Virion, Illumina 15 ATGTCA)
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
Library generated from total RNA immunoprecipitated with nonspecific polyclonal sera, and fragmented with RNAse T1
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| Data processing |
Libraries were demultiplex and trimmed to remove adaptors on the MiSeq interface to generate the raw sequence files.
All analyses of data sets were performed using the Galaxy web services found at Usegalaxy.org, where applicable the programs utilized are indicated by "_" and the version is noted by the (_) following immediately after. Deviations from the default settings are noted.
Library quality was assessed using "FastQC"
Fastq files were converted to Fasta via the "fastQ_to_fasta" tool.
The cDNA libraries were aligned to the SINV Toto1101 genome via "LastZ" (1.2.2) with the following parameters- Search Plus sense only, Require 22bp word with matches in 14 specific positions, allow 2 transitions per hit, Gap open penalty= 1000, Gap Extension. Penalty= 1000,
LastZ output files were converted from SAM format to BAM format using "SAM-to-BAM" (2.1.1) using the SINV Toto1101 Reference Genome.
Depth of coverage was determined using SAMTools to generate a pileup file for each library via "Generate Pileup" (1.2.2).
Base representation, identity, and coverage was determined via the filtering of the pileup data using "Filter Pileup" (1.0.2) to report coverage at each nucleotide.
Tabular data from the filtered pileup files was imported into Excel and analyzed via subtractive analysis to determine regions of significant enrichment for each cDNA library.
Genome_build: SINV Toto1101 Genomic Sequence
Supplementary_files_format_and_content: Single Processed Data File, in .xlsx format. This file contains summary information for the analysis, the depth of coverage at a nucleotide level of resolution for each cDNA library (on different tabs), and the subtractive analysis used to identify the sites of interaction is included on a separate tab.
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| Submission date |
Jun 09, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Kevin J Sokoloski |
| Organization name |
University of Louisville
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| Department |
Microbiology and Immunology
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| Lab |
Sokoloski Lab
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| Street address |
505 S. Hancock St.
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| City |
Louisville |
| State/province |
KY |
| ZIP/Postal code |
40202 |
| Country |
USA |
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| Platform ID |
GPL15520 |
| Series (1) |
| GSE99879 |
Identification of Interactions between Sindbis Virus Capsid Protein and Cytoplasmic vRNA as Novel Virulence Determinants |
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| Relations |
| BioSample |
SAMN07210870 |
| SRA |
SRX2899927 |
| 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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