4.2. Chromatin Immunoprecipitation, RNA Isolation, Reverse Transcription PCR, and qPCR

CNSs were dissected in ice cold PBS from 350–600 third instar larvae of each genotype per biological replicate. Dissected CNSs were placed in 1× PBS and stored at −80 °C. Chromatin was sheared using a Tissue Chromatin Shearing Kit with SDS Shearing Buffer (Covaris). Dissected CNSs were washed twice with 1× PBS, fixed in Buffer A with 1% methanol-free formaldehyde at room temperature for five min, and then Quenching Buffer E was applied to stop the fixation. The tissue was centrifuged at 4 °C for five min, after which the supernatant was removed. The pelleted tissue was washed twice with ice cold 1× PBS. The Wash buffer (WB) was removed and then the tissue was homogenized for 2–3 min in 500 µL Lysis Buffer B. The latter was subsequently added to increase the volume to 1 mL, followed by rocking incubation at 4 °C for 20 min 3 s of vortexing every 10 min. Lysed tissue was next pelleted, resuspended in WB C, washed, and resuspended in Covaris SDS Shearing Buffer D, which remained on the tissue for 10 min with occasional vortexing. Chromatin was sheared after transfer to a TC 12 × 12 tube for 10 min by a Covaris E220 Ultrasonicator. Sheared chromatin was visualized on an agarose gel containing 1.5% Ethidium Bromide (Fisher Scientific, Waltham, MA, USA) to confirm 100–600 bp chromatin fragments. Chromatin was then immunoprecipitated using a Magna ChIP HiSens Kit (Millipore Sigma, Burlington, MA, USA). Then, 50 µL of sheared chromatin was incubated for three hours with coated magnetic beads bound with either rabbit α-GFP (Abcam, ab290) or rabbit α-IgG (Abcam, ab171870). Chromatin was then eluted from the magnetic beads and incubated in RNase A (10 mg/mL, Fisher Scientific), for 30 min followed by incubation at 57 °C overnight in Proteinase K (10 mg/mL, Millipore). The next day, the Proteinase K was inactivated by incubating for 15 min at 75 °C. The QIAquick PCR Purification Kit (Qiagen, Germantown, MD, USA) was used to isolate DNA, which was then stored at −20 °C for qPCR.

RNA was isolated from third instar larval CNSs or muscle pelts, which were dissected from males and females. Dissections were performed in Roger’s Ringer solution (135 mM NaCl, 5 mM KCl, 4 mM MgCl₂*6H₂O, 1.8 mM CaCl₂*2H₂O, 5 mM TES, 72 mM Sucrose, 2 mM glutamate, pH 7.15). CNSs and muscle pelts were placed in nuclease-free 1.5 mL centrifuge tubes containing Invitrogen RNAlater Stabilization Solution (Fisher Scientific AM7020) and stored at −20 °C. RNA was isolated using the Invitrogen Purelink RNA Mini Kit (Fisher Scientific 12-183-025). RNA concentrations were obtained using an Implen Nanophotometer N50. Each technical replicate included 30 CNSs or eight muscle pelts per genotype. Two to three technical replicates were used to calculate relative fold changes.

QPCR Primers were designed using PerlPrimer (v. 1.1.21). RT-qPCR was performed using the iTaq Universal SYBR Green One Step Kit (Bio-Rad, 1725151, Hercules, CA, USA) and a CFX Connect Real-Time PCR Detection System (Bio-Rad). Here, 100 ng of RNA was used for each reaction. Two to three biological replicates each including three technical replicates were used for data analyses. 2^−ΔΔC(t) values [107] were determined by calculating the difference between the C(t) value of the target transcript reaction and the C(t) value for GAPDH to obtain ΔC(t) for each transcript. Next, the difference between the control and kis mutant ΔC(t)s was calculated and log transformed to obtain the 2^−ΔΔC(t).