Computational protocol: Genome-wide features of neuroendocrine regulation in Drosophila by the basic helix-loop-helix transcription factor DIMMED

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Protocol publication

[…] At the end of sorting, cells were transferred directly into Qiagen's RLT buffer supplemented with beta-mercaptoethanol (Qiagen RNA MinElute kit). Most investigators harvesting nano scale RNA samples for microarray gene expression profiling use the Arcturus PicoPure kit (,). This kit contains a poly(dI:dC)-based proprietary nucleic acid carrier embedded in the column. This carrier does not interfere with microarray applications, but could interfere with deep sequencing of RNAs isolated this way. Therefore, we decided to use the Qiagen kit for small samples, which lacks carriers. After transferring cells into RLT buffer, they were vortexed for 6 s, and lysed by passing the suspension five times through a 21.5 gage needle mounted on a 3-ml syringe. RNA was isolated as recommended by Qiagen's MinElute protocol, with the exception of using 65°C-heated water for enhanced RNA recovery. Next, DNA in the sample was digested with the DNase I Turbo DNA-free kit (Ambion). After DNA removal, RNAs isolated from GFP+ and GFP− cells from each of the two experiments were pooled.Samples with pooled GFP+ and GFP− RNAs were then submitted to the Genome Technology Access Center (Washington University Genetics Department). RNA quality was then checked by Qubit (Invitrogen), NanoDrop (GE Health) and Bioanalyzer (Agilent) quality control assays. Neither sample showed signs of degradation or DNA contamination. Thirty-five nanograms of RNA from GFP+ cells left and 35 ng of RNA from GFP− cells were processed for sequencing on Illumina's HiSeq 2000 platform.Because Illumina's deep sequencing protocols require 10 μg of RNA, the samples were amplified with the NuGen Ovation RNA-Seq system, a single primer-based RNA amplification product that uses isothermal amplification (,). After amplification, the samples were prepared for deep sequencing according to standard Illumina procedures, which included a poly-A selection step and multiplexing. Sequencing was performed in a single lane of an Illumina HiSeq 2000 machine. Base calls were made by Illumina's software (Eland), followed by demultiplexing.Cufflinks and Tophat algorithm were used to align raw sequence reads to the reference genome and to map splice junctions against Drosophila dm3 r5.50 genome release (,). Alignments were indexed and sorted for visualization in the Integrated Genomics Viewer (). Indexed RNA-Seq reads were visualized as coverage tracks. In order to be able to compare samples directly, coverage tracks were always graphed with identical y-axis coordinates. […]

Pipeline specifications

Software tools ELAND, Cufflinks, TopHat
Application RNA-seq analysis
Organisms Drosophila melanogaster