EC-tagging for RNA purification and RT-qPCR
5-ethynylcytosine was synthesized as previously described [20].
Larvae were reared at 25°C and fed 1 mM 5EC from 48 – 72 hours after
hatching. Total RNA was extracted from crudely dissected central nervous
system tissue using Trizol. 10 µg of RNA was biotinylated using Click-iT
Nascent RNA Capture reagents (ThermoFisher) and purified on Dynabeads
MyOne Streptavidin T1 magnetic beads (ThermoFisher) as previously
described [20]. After the final wash, beads containing captured RNA
were used to make first-strand cDNA with the SuperScript VILO cDNA
Synthesis Kit (Invitrogen), as previously described [20]. Real-time
PCR quantitation was performed on a Rotor-Gene Q (Qiagen) in 20 µL
reactions using SYBR green detection. Pre-designed QuantiTect primers
(Qiagen) were used for Syt1 PCR. RNA polymerase II subunit B
(Polr2B ) primers, used for normalization, were Forward primer:
TCAGCGTCTTAAGCACATGG and Reverse primer: TCGGAGACCTCGAATAAACG.
Previously described sequences were used for pre-rRNA specific primers
[26], synthesized by Integrated DNA Technologies. Pre-rRNA andSyt1 Ct values were normalized to Polr2B and
relative abundance calculated by the equation, fold
change = 2-Δ(ΔCt ). RT-qPCR was performed on
biological replicates of each genotype.