Qx200 evagreen digital pcr supermix

Total RNA was separately extracted from three control and three Lhx4 null mice at adult with RNeasy Mini Kits (QIAGEN, Valencia, CA) in accordance with the manufacturer’s protocol. cDNA was synthesized from total RNA using iScript cDNA Synthesis Kit (Bio-Rad). ddPCR was performed by using QX200 Droplet Digital Systems (Bio-Rad). QX200 EvaGreen Digital PCR Supermix (Bio-Rad) was used for the ddPCR reaction. The analysis was performed on the QuantaSoft Analysis Pro (Bio-Rad). The housekeeping gene Actb was used for internal control.

All dPCR assays were carried out on the QX200™ Droplet Digital™ PCR System (Bio-Rad) using the QX200 EvaGreen Digital PCR Supermix (Bio-Rad) according to the manufacturer’s instructions. Primers and thermocycling conditions for all dPCR assays are listed in Supplemental S8.

After cell lysis in TRI Reagent (ThermoFisher), Direct-zol RNA MiniPrep kit (ZymoResearch) or Total RNA Zol-Out kit (A&A Biotechnology) was used for total RNA isolation. For Flp-In T-REx-293 cell lines, a fraction of lysates prepared in Cytoplasmic Lysis Buffer [PBS, 0.1% NP40, cOmplete EDTA-free Protease Inhibitor Cocktail (Roche)] was mixed with four volumes of TRI Reagent for further isolation. The concentration of isolated total RNA was assessed by measurement at 260 nm using DeNovix spectrophotometer. Reverse transcription was performed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and random hexamer primers (Promega), according to the manufacturer's protocols. RT-qPCR was performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and CFX Connect Real-Time System (Bio-Rad), according to the manufacturer's protocols and established guidelines for qPCR. Digital droplet PCRs (ddPCRs) were prepared using DG8 cartridges and gaskets, QX200 Droplet Generation Oil and QX200 EvaGreen Digital PCR Supermix (BioRad) and performed on QX200 Droplet Digital PCR System (BioRad), according to the manufacturer's protocols. All primer sequences are listed in Table S4.