Reference
Citation
Brown, J.B., Boley, N., Eisman, R., May, G.E., Stoiber, M.H., Duff, M.O., Booth, B.W., Wen, J., Park, S., Suzuki, A.M., Wan, K.H., Yu, C., Zhang, D., Carlson, J.W., Cherbas, L., Eads, B.D., Miller, D., Mockaitis, K., Roberts, J., Davis, C.A., Frise, E., Hammonds, A.S., Olson, S., Shenker, S., Sturgill, D., Samsonova, A.A., Weiszmann, R., Robinson, G., Hernandez, J., Andrews, J., Bickel, P.J., Carninci, P., Cherbas, P., Gingeras, T.R., Hoskins, R.A., Kaufman, T.C., Lai, E.C., Oliver, B., Perrimon, N., Graveley, B.R., Celniker, S.E. (2014.8.27). modENCODE RNA-Seq data remapped to BDGP Release 6 genome assembly.  Nature
FlyBase ID
FBrf0226107
Publication Type
Personal communication to FlyBase
Abstract
PubMed ID
PubMed Central ID
Text of Personal Communication
The files for the RNA-seq alignments to Release 6 are available. These files contain only the uniquely aligned sequences. The spreadsheet listing each sample that was aligned to Release 6, along with its RNA ID, BS ID, read length, aligned read count, SRA and DCC IDs. For experiments of mixed read length, the spreadsheet specifies the number of reads of each length.
The protocol used for the alignments is as follows:
- Ran the STAR aligner v2.3.0e (Linux x86_64) with default parameters on the FASTQ files to generate multiply-mapped BAM files.
- URL for STAR aligner is https://code.google.com/p/rna-star/
- Used the “samtools sort” command to sort the BAM files.
- Filtered the multiply-mapped BAM files to only include reads with only 1 aligned hit ( NH:i:1  attribute) to generate uniquely-mapped BAM files.
- Ran a custom script to convert BAM files into bedgraph files (bam2bedgraph.cc).
- Ran the UCSC bedGraphToBigWig tool to convert bedgraph files into bigwig files.
- Used the “samtools flagstat” command to count the number of aligned reads in each uniquely mapped BAM file.
In addition to the published samples there are six new treatment samples, as listed below:
Resveratrol_Treatment_100uM_10_Day_Adults.bam
Rotenone_Treatment_Starved_10_D_Adult.bam
Virus_Sindbis_Female.bam
Virus_Sindbis_Larvae.bam
Virus_Sindbis_Male.bam
Virus_Sindbis_Pupae.bam
All the metadata for the published treatments can be found in Brown et al., 2014 Diversity and dynamics of the Drosophila transcriptome. Nature 512(7515):393-399. Since the Sindbis virus treatments were completed after the modENCODE DCC freeze and therefore not included in the manuscript, the metadata for the viral samples is as follows. A transgenic stock was obtained from Dr. Richard Hardy, Indiana University, Bloomington. This strain contained two transgenic elements. The first pUAST- SINrep:GFP  is a construct in the pUAST vector containing pSINrep/GFP that encodes the non-structural proteins of the Sindbis virus tagged with GFP preceded by 5 UAS sequences. The GFP tag marks the production of Sindbis viral RNA but the RNA is incapable of producing capsid protein and therefore no mature virus van be made by the flies expressing this transgene. The second is a standard  Act5C::Gal4  driver (P{Act5C-GAL4}17bFO1) obtained from the Bloomington Drosophila Stock Center. Both inserts are on the third chromosome and in the original stock the chromosome is homozygous lethal and balanced over TM3, Sb1. Males from this stock were crossed to Oregon R virgin females and progeny Sb+ adult males expressing GFP collected. These were crossed to Oregon R virgin females and the GFP positive male progeny collected. This procedure was continued for 10 further generations in order to place the  Act5::Gal4=>UAS::SIN  system into the same genetic background as previously used for RNA extraction. After the 12th generation adult male and female progeny expressing GFP were collected, aged for four days and were flash frozen in liquid nitrogen. GFP positive larvae and pupae (each containing mixed ages and sexes) were also collected and were flash frozen in liquid nitrogen for RNA preparation. The genotypes of the starting stock and the characterization of the transgenic lines is described in Avadhanula et al., 2009. A novel system for the launch of alphavirus RNA synthesis reveals a role for the Imd pathway in arthropod antiviral response. PLoS Pathog. 5(9): e1000582.
Due to the unavailability of Illumina reagents at the time of library construction, the libraries for these six additional treatment samples were prepared differently than the other treatment samples; libraries were derived from poly(A)+ selected mRNA using a dUTP-based protocol in the Gingeras Lab at CSH. While RNA-Seq for most treatment samples is 99.97% to 99.9% stranded data, the dUTP libraries are only 90% to 97% stranded. There was no evidence that the larvae or adults ingested the rotenone as there was no green die in the gut. The only perturbation to the transcriptome is assumed to be from starvation.
DOI
Related Publication(s)
Research paper
Diversity and dynamics of the Drosophila transcriptome.
Brown et al., 2014, Nature 512(7515): 393--399 [FBrf0225793]
The developmental transcriptome of Drosophila melanogaster.
Graveley et al., 2011, Nature 471(7339): 473--479 [FBrf0213330]
Associated Information
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Associated Files
File date: 2014.8.27 ; File size: 27376 ; File format: xlsx ; File name: Celniker.2014.8.27.Metadata_modENCODE_RNA-Seq_Release6_Remapped.xlsx
Other Information
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    Language of Publication
    English
    Additional Languages of Abstract
    Parent Publication
    Publication Type
    Journal
    Abbreviation
    Nature
    Title
    Nature
    Publication Year
    1869-
    ISBN/ISSN
    0028-0836
    Data From Reference
    Datasets (127)