Rq1 buffer

RNA was extracted from approximately 30–50μl of fly heads using 3X volume TRI Reagent (Sigma-Aldrich, St. Louis, MO). 1/5 volume of 100% chloroform (Sigma-Aldrich) was added and incubated at room temperature for 10 minutes. Upper aqueous layer was recovered after spinning down at 13,000 rpm for 15 minutes. Same volume of 100% isopropanol was added and incubated at −20°C overnight to precipitate RNA. After spinning down, RNA pellet was washed with 200μl 70% ethanol once, resuspended in 20μl 1X RQ1 buffer (Promega, Madison, WI), and treated with 2μl RQ1 DNase (Promega) at 37°C for 30 minutes prior to the incubation with 2μl RQ1 DNase stop solution (Promega) at 65°C for 10 minutes. cDNA was generated from equal amount of RNA for each sample using Superscript IV (Thermo Fisher Scientific, Waltham, MA). Real-time PCR was performed using SsoAdvanced SYBR green supermix (Bio-Rad, Hercules, CA) in a CFX96 or CFX384 (Bio-Rad). Three technical replicates were performed for each of three biological qPCR replicates.
To confirm expression of clk isoforms, RNA was extracted as above. cDNA was generated using gene-specific primer clk(698R). PCR was then performed using clk(1F) and clk(101R) prior to resolving PCR products in 2% TBE gel. Bands were excised, and gel extracted for Sanger sequencing. Sequences for all primers are presented in Table S5.