Qscript cdna supermix

RNA from iNeurons cultured were extracted using the RNeasy Mini kit (Qiagen). cDNA was generated using the qScript cDNA SuperMix (QuantaBio). ddPCR was performed as previously described (Deneault et al., 2018 (link)). Quantification was normalized to the control of each condition and run along a no template control. The data was analyzed and produced on the QuantaSoft software (Bio-Rad). The following assays were used: SCN2A exon 4–5 (Hs01109883_m1, ThermoFisher) and TBP (Hs00427620_m1, ThermoFisher).

Complementary DNA was synthesized from total RNA using qScript cDNA SuperMix (Quantabio, Beverly, MA, USA) according to the supplier’s recommendations. For quantitative PCR (qPCR), reactions were performed in triplicate using SYBR Green PCR Master Mix (Applied Biosystems, Life Technologies, Warrington, Florida, USA) in an ABI Prism 7900 Sequence Detection system (Applied Biosystems, Life Technologies, Singapore). Two genes were used as internal controls: TATA-BOX binding protein (tbp) and eukaryotic elongation factor-1 (eef1). Primers for the genes of interest were designed using NCBI primer design tool. Primer sequences are listed in Supplementary Table 1. Gene expression was analyzed with the qBase 1.3.5 software using the comparative cycle threshold method.

To track the expression of tra genes in the adherent AIEC population, wild-type AIEC was grown in LB at 37 °C overnight. The next day, bacteria were used to either infect Caco-2 cells at an MOI 1000:1 or cell-free DMEM media for 4 h. Importantly, non-adherent bacteria were washed out with sterile PBS. At the end of the infection, bacterial pellets were harvested from both conditions and nucleic acid was extracted using the Trizol-based method. Upon treatment with RNase-free DNase (Thermo Fisher), cDNA was obtained using either qScript cDNA Supermix (Quantabio) or protoscript (NEB). qPCR was performed with SYBR green SuperMix (Quanta Biosciences) on the LightCycler 480 (Roche) or Bio-Rad CFX96 real-time PCR detection systems. Primers used are listed in Supplementary Data 2. Data analysis was performed using the LightCycler software (version 1.5). Gene expression was normalized to the housekeeping gene, gyrB.

Total RNA was extracted from the hypothalamus using Qiagen RNeasy Mini kits (Qiagen, Germantown, MD, USA) and reverse transcribed using qScript cDNA Supermix (Quantabio, Beverly, MA, USA). Gene expression was assessed using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) and TaqMan probes (Gapdh: Mm99999915_g1, Hprt: Mm03024075_m1, Ghsr: Mm00616415_m1, Hcrtr1: Mm01185776_m1, Npy: Mm01410146_m1, Pomc: Mm00435874_m1, Htr2c: Mm00434127_m1). Results are expressed at 2^-ΔCt normalized to the geometric mean of the Ct value of Gapdh and Hprt. The geometric mean of Gapdh and Hprt were not significantly different between treatment groups.

MDM sorted as described above in ‘Flow cytometry’ were collected into tubes containing RLT buffer (Qiagen, Hilden, Germany) and RNA was isolated using RNeasy Kit (Qiagen) with on-column DNase I digestion. RNA was reverse transcribed using qScript cDNA SuperMix (Cat #95048, Quantabio, Beverly, Massachusetts). Quantitative PCR was performed using TaqMan Gene Expression MasterMix (ThermoFisher, cat# 4369016) on an Applied Biosystems 7300 Real-Time PCR System using TaqMan Gene Expression primers with FAM-MGB probe. The primer/probe sets for ACTB (Hs99999903), MRC1 (Hs00267207), POL2A (Hs02786624), and GAPDH (Hs00172187) were purchased from ThermoFisher. Reactions were quantified using ABI Sequence Detection software compared to serial dilutions of cDNA from mock-treated cells. Measured values for all genes were normalized to measured values of GAPDH or ACTB as indicated.