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General Information
Name
mE_mRNA_P5
Species
D. melanogaster
Result type
FlyBase ID
FBlc0000105
Project
Data Provider
Title
RNA-Seq coverage profile of D. melanogaster, iso-1 strain, pupa (12hr APF), modENCODE, unstranded.
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
    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).

    Note that for each pair of paired-end reads, the two reads were mapped independently, and only those reads mapping uniquely to the genome were included in the data submission to FlyBase. In other words, information from one read was not used to resolve ambiguous mapping of its paired read.

    Comments
    Associated Data
    Size
    Uniquely aligned reads; as reported.
    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 (10)
    Reported As
    Name Synonyms
    RNA-Seq coverage profile of D. melanogaster, iso-1 strain, pupa (12hr APF), modENCODE, unstranded.
    Secondary FlyBase IDs
      References (8)