2016-10-26T17:58:25Z (GMT) by Carl S. Thummel William E. Barry
Total RNA was extracted from dHNF4 mutants and genetically-matched control adult males (8 days old) using TriPure isolation reagent (Roche) followed by further purification/cleanup by RNeasy kit (QIAGEN). RNA from 4 biological replicates (n=40 flies per sample) per genotype was polyA-selected and assembled into barcoded libraries and pooled into a single-flow cell lane for Illumina HighSeq2000 50-cycle single-read sequencing, which produced ≥ 21.9 million reads per sample. Standard replicate RNAseq analysis was performed using USeq and DESeq analysis packages with alignment to the Drosophila melanogaster dm3 genome assembly. Transcripts meeting a cutoff of ≥1.5 fold difference in mRNA abundance (~Log2 ≥ +/- 0.6) and FDR ≤ 1% were considered as differentially expressed genes. RNA quality control, library preparation, sequencing, and data analysis were performed at the University of Utah High Throughput Genomics and Bioinformatics Core Facilities.