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General Information
Name
mE_mRNA_L_Sindbis_virus
Species
D. melanogaster
Result type
FlyBase ID
FBlc0000675
Project
Data Provider
Title
RNA-Seq coverage profile of D. melanogaster, Oregon-R-modENCODE strain, larva, Sindbis virus treatment.
Status
Current
Accessions
    Biosample Source
    Overview
    Sex
    Tissue isolated
    Other tissues studied
    Cell component
    Cell line
    Key genes
    Sample preparation

    A transgenic stock carrying the constructs P{Act5C-GAL4} and P{UAS-SINrep.GFP} on the same chromosome was used to constitutively express non-structural components of the Sindbis virus (see FBrf0208818). This stock was crossed to Oregon R virgin females, and males expressing the GFP-tagged Sindbis proteins were selected for breeding. This procedure was continued for 10 further generations to place the transgenes into the same genetic background as other treatment experiments. Larvae (mixed stage and sex) were collected after the 12th generation and flash frozen in liquid nitrogen for RNA preparation.

    Biosamples analyzed by this result (1)
    Biosample
    Type
    Title
    D. melanogaster, Oregon-R-modENCODE strain, larva, Sindbis virus treatment, source for RNA.
    Data Analyzed
    Key genes
    Protocol

    For the Sindbis virus, resveratrol and rotenone/starved treatment samples, the RNA-Seq libraries were generated using a dUTP-based method (PMID: 22955620) that gives 90% to 97% stranded data. The Illumina kit used for the other treatment samples give 99% stranded data.

    Total RNA was isolated using Trizol, then Qiagen RNeasy spin columns. mRNA was isolated using Dynal oligo(dT) beads, then fragmented using divalent cations under elevated temperature, followed by sequential ligation of RNA linkers to the 5’ and 3’ ends. Next, reverse transcription was performed using a primer complementary to the 3’ linker and PCR was performed using primers complementary to both linkers. ~ 300 bp fragments were isolated from an agarose gel and gel-purified again.

    Mode of Assay

    Read length (bases):101

    The samples were quantitated using a Nanodrop, and loaded onto a flow cell for cluster generation and sequenced on an Illumina Genome Analyzer II using either single read or paired end protocols (Illumina).

    Raw Data Analyzed (1)
    Assay / Reagent collection
    Type
    Title
    RNA-Seq of D. melanogaster, Oregon-R-modENCODE strain, larva, Sindbis virus treatment.
    Processed Data Analyzed (0)
    Result
    Type
    Title
    Analysis
    Reference Genome
    Reference Annotation
    Data analysis

    Reads were aligned to Dmel_Release_6 using the STAR aligner v2.3.0e (Linux x86_64) with default parameters on the FASTQ files to generate multiply-mapped BAM files. These were filtered to include reads with only 1 aligned hit ( NH:i:1 attribute) to generate uniquely-mapped BAM files. A custom script was used to convert BAM files into bedgraph files (bam2bedgraph.cc).

    Comments
    Associated Data
    Size
    386,953,500 Number of unique reads.
    Files
    Additional Information

    The RNA-seq profiles displayed by FlyBase in GBrowse and used for RPKM calculation can be accessed at the FTP link below as .wig files. Please take note of how these FlyBase .wig files represent data for a contiguous sequence of bases with the same signal value. The value is declared only for the first position of that region, and applies to all positions that follow (these are not explicitly listed) until a new value at a new base position is declared.

    Synonyms and Secondary IDs (5)
    Reported As
    Symbol Synonym
    Cherbas_796 Cherbas_797
    Virus_Sindbis_Larvae
    mE_mRNA_L_Sindbis_virus
    Name Synonyms
    RNA-Seq coverage profile of D. melanogaster, Oregon-R-modENCODE strain, larva, Sindbis virus treatment.
    Secondary FlyBase IDs
      References (2)