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General Information
Name
mE_mRNA_P5_junctions
Species
D. melanogaster
Result type
FlyBase ID
FBlc0000142
Project
Data Provider
Title
RNA-Seq exon junctions in D. melanogaster, iso-1 strain, pupa (12hr APF), modENCODE.
Status
Current
Accessions
    Biosample Source
    Overview
    Sex
    Other tissues studied
    Cell component
    Cell line
    Key genes
    Methods
    Sample preparation

    Fly stocks were reared in bottles at 24oC and after about 7 days, white prepupa on bottle walls were marked on the overlying glass and allowed to age as appropriate. Individuals were then transferred to a tube on dry ice.

    Biosamples analyzed by this result (1)
    Biosample
    Type
    Title
    D. melanogaster, iso-1 strain, pupa (12hr APF), source for RNA.
    Data Analyzed
    Key genes
    Protocol

    Frozen samples were homogenized and extracted using the TRIzol reagent protocol (Invitrogen). RNA was purified on an RNeasy spin column (Qiagen), and DNase treated. Polyadenylated RNAs were purified from total RNA extracts via oligo(dT) binding, using standard Illumina protocol. The poly(A)+ RNA was fragmented using divalent cations under elevated temperature, following by first and second strand cDNA synthesis primed with random hexamers. The cDNA fragments were end-repaired using T4 DNA polymerase and Klenow DNA polymerase, and phosphorylated at their 5' ends with T4 polynucleotide kinase. After adding A bases to the 3' end of the DNA fragments, Illumina adaptor oligonucleotides were ligated to the ends and ~ 300 bp fragments were isolated from an agarose gel, enriched by PCR amplification, and gel-purified again.

    Mode of Assay

    Read length (bases):76

    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, iso-1 strain, pupa (12hr APF), modENCODE, unstranded.
    Processed Data Analyzed (0)
    Result
    Type
    Title
    Analysis
    Methods
    Reference Genome
    Reference Annotation
    Data analysis

    RNA-Seq reads were aligned to databases of predicted splice junctions and to gene models using TopHat, STAR and BLAT. A number of criteria were used to distinguish true splice junctions from false positives. First, only reads with a minimum of 6nt overhang across a junction were considered. Second, an entropy score (which reflects the number of offset reads) was calculated for each splice junction, and only junctions with entropy score >2 in at least two biological samples were considered. The set of junctions was filtered further to exclude junctions when intron length less than 41 nt, junctions that join parologous genes and non-canonical splice junctions not supported by other experimental support.

    Comments
    Associated Data
    Size
    Files
    Additional Information
    Synonyms and Secondary IDs (8)
    Reported As
    Symbol Synonym
    BS133
    WPP +12hr
    mE_mRNA_P5_junctions
    Name Synonyms
    RNA-Seq exon junctions in D. melanogaster, iso-1 strain, pupa (12hr APF), modENCODE.
    Secondary FlyBase IDs
      References (3)