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Protocol publication

[…] Total RNA from pooled third instar larval wing imaginal discs (1000 pairs per sample preparation) expressing UAS-lacZ or UAS-tsu RNAi (VDRC#107385) driven by T80-Gal4 was extracted in duplicates using TRIzol. Poly(A)+ mRNAs were enriched using Dynabeads oligo (dT) beads (NEB). RT reactions and purification of cDNA templates were performed following the RNA-seq sample preparation protocol from Illumina. Each cDNA sample was sequenced on an IlluminaHiseq 2500 system.Whole transcriptome reads were aligned using the TopHat (v2.0.13) () with the Ensembl Drosophila_melanogaster.BDGP5.78.gtf as a reference ( In total, 22,563,753 and 24,660,199 of 125 bp reads pairs for duplicated tsu samples and 22,610,210 and 28,651,178 of 125 bp reads pairs for lacZ samples were sequenced, respectively. The transcription analysis was performed using Cufflinks (v2.2.1) (). DEXseq was used to plot the transcripts to each gene (). RPKM method (reads per kilobase of transcript per million mapped sequence reads; ) was used for normalizing gene counts. We calculated the ratio of RPKM between the tsu and lacZ samples. The R density plot package (, R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. was used to generate the distribution plot shown in . The Seq data was deposited to NCBI website: […]

Pipeline specifications

Software tools TopHat, Cufflinks, DEXSeq
Databases BDGP
Application RNA-seq analysis
Organisms Drosophila melanogaster, Homo sapiens