Hiscript 2 reverse transcriptase supermix

To further determine the transcriptional level of target genes (ituD, ituA, ituB, ituC, degQ, degU, comA, glnR, sfp, codY, abrB and yczE), bacterial cultures were harvested after 24 h incubation. Total RNAs of these bacterial cultures were isolated using the commercial RNApure Bacteria kit (DNase I) (Cwbio, Beijing, China). Then, cDNA was synthesized using the extracted RNA samples and HiScript^® II Reverse Transcriptase SuperMix (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Real-time PCR was carried out using FastStart Universal SYBR Green Master (Roche, Basel, Switzerland) on a StepOnePlus™ real-time PCR system (Applied Biosystems, Foster City, CA, USA). PCR conditions were as follows: pre-incubation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s and extension at 72 °C for 20 s. The transcriptional levels of target genes were normalized against that of rspU [50 (link)].

Fifteen brains, 15 midguts, or five adult flies from each genotype and αTC1 cells were lysed to extract total RNA using Trizol (Invitrogen). cDNA synthesis was performed using HiScript II reverse transcriptase supermix (R223-01, Vazyme). Quantitative real-time PCR was performed using ChamQ SYBR master mix (Q311-02, Vazyme) on a CFX384 Real-Time System/C1000 Thermal Cycler (BioRad). Fly and mouse gene expressions were normalized to RpL32 and β-actin, respectively. qPCR primers used in this study are listed in Supplementary Table S4.