Regulation of fiber-specific actin expression by the Drosophila SRF ortholog Blistered

Gene expression in the indirect flight muscles was evaluated by Illumina next-gen sequencing under normal conditions and in response to a genetic knockdown of Blistered (bs), a fly homolog of Serum Response Factor. Overall design: Progeny of the following crosses were used: 1151-Gal4 x w1118 (control) and 1151-Gal4 x bs[KK108659] (bs knockdown). For each sample (either control or knockdown), indirect flight muscles (IFMs) were dissected from 4 young females in 1M sucrose and immediately dissolved in the solubilizing buffer provided by RNeasy RNA purification kit (Qiagen). Total RNA was extracted following the kit manufacturer's protocol, including 15-min on-column DNAse I digestion. 100-200 ng of purified total RNA were submitted for sequencing at the Sulzberger Genome Center (Columbia University, New York). At the Center mRNA transcripts were enriched by poly-A pull-down using TrueSeq RNA prep kit (Illumina) and ~30 millions of single-end, 100-bp reads were produced per each sample by Illumina HiSeq2000 instrument. The post-run analysis was performed on the web-based platform Galaxy (usegalaxy.org). Transcript quantification was done by Salmon tool (doi:10.1101/021592) that used reference transcriptome and gene coordinates from D. melanogaster 6.21 genome release. Comparative analysis between control and knockdown samples was done by DEseq2 (doi:10.1186/s13059-014-0550-8).