*2.13. Microarray*

Total RNA was isolated from 3-week-old female wild type and *slo* mutant flies using Qiagen RNAeasy kit. RNA was treated with RNase-free DNAse I. RNA was quantified on a Nanodrop

ND-100 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), followed by RNA quality assessment by analysis on an Agilent 2100 bioanalyzer (Agilent, Palo Alto, CA, USA). Fragmented biotin-labeled cDNA (from 100 ng RNA) were prepared using the GeneChip WT Plus kit.

Each <sup>A</sup>ffymetrix gene chip*Drosophila* array (Affymetrix, Santa Clara, CA, USA) was hybridized with the fragmented and biotin-labeled target (4.5 μg) in 200 μL of hybridization cocktail. Target denaturation was performed at 99 ◦C for 2 min and then 45 ◦C for 5 min, followed by hybridization for 18 h. Then the arrays were washed and stained using GeneChip Fluidic Station 450, and hybridization signals were amplified using antibody amplification with goa<sup>t</sup> IgG (Sigma-Aldrich, St. Louis, MO, USA) and anti-streptavidin biotinylated antibody (Vector Laboratories, Burlingame, CA, USA). The chips were scanned on an <sup>A</sup>ffymetrix Gene Chip Scanner 3000, using Command Console Software. Background correction and normalization were done using Robust Multichip Average with Genespring V 14.9 software (Agilent). A 1.5-fold differentially expressed gene (*p* ≤ 0.05 values) list was generated. The listing of differentially expressed genes and their fold change were loaded into Ingenuity Pathway Analysis (IPA) 5.0 software (Qiagen Inc., https://www.qiagenbioinformatics.com/products/ingenuitypathway-analysis) to perform biological network and functional analyses. IPA converts gene sets (with or without expression information) into related molecular networks based on IPA knowledge database. Core analysis was performed for and the genes were categorized based on molecular function, mapped to genetic networks, and ranked by score. The score reflects the probability that a collection of genes equal to or greater than a number in the network could not be achieved by chance alone. A score of more than 10 was used as a cutoff for identifying specific gene networks (n = 3 independent experiments with RNA isolated from 100 flies each).
