Transcriptome comparison of wild-tyle (w1118) vs. actβ mutants (act80/act4E) Drosophila larvae

TGFβ/Activin subfamilies of ligands. In Drosophila the TGFβ/Activin branch includes three ligands, Myoglianin (Myo), Activin-β (Actβ) and Dawdle (Daw). All three ligands signal through the same type I receptor, Baboon, in conjunction with a type II receptor, Punt or Wit. Baboon has three distinct splice isoforms, termed Babo A, Babo B and Babo C, that differ only in exon 4, which encodes the ligand binding domain. This difference enables each ligand to potentially signal through a single splice isoform39. Formation of the ligand receptor complex stimulates phosphorylation of the receptor-Smad, dSmad2/Smox, which translocates to the nucleus as a complex with the co-Smad Medea where it serves as a transcriptional transducer. In order to gain insight into the genes regulated by Actβ we carried out a transcriptomic analysis comparing Actβ mutants to wild-type controls. Animals were raised on apple juice plates supplemented with yeast paste until the early L3 time point (approximately 68 hours AEL). RNA was extracted 8 larvae of each biological repliacte and a minimum of five biological replicates were submitted for each genotype: wild-type control (w1118) and actβ mutants (act80/act4E). The Actβ80 allele is an EMS-induced substitution leading to a premature stop codon and presumed to be a null mutation. Actβ4E was generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Two guide RNAs were cloned into the BbsI site of the pU6-BbsI-chiRNA plasmid (obtained from Addgene) and injected by Best Gene into w1118; PBac{y[+mDint2]=vas-Cas9}VK00027 on chromosome 3 (#51324; BDSC).Total RNA (3 μg per sample) was submitted to the University of Minnesota Genomics Center (UMGC) for quality assessment and Illumina next-generation sequencing. 2 x 150bp fastq.gz paired-end reads for samples (n=30.3 Million average reads per sample) were trimmed using Trimmomatic (v 0.33) enabled with the “-q” option; 3bp sliding-window trimming from 3’ end requiring minimum Q30. Quality control on raw sequence data for each sample was performed with fastq.gzC. Read mapping was performed via Hisat2 (v2.1.0) using the Drosophila genome (Drosophila melanogaster (BDGP6.28) as a reference. Gene quantification was done via Feature Counts for raw read counts.