Maxima first cdna synthesis kit

RNA was extracted with phenol-chloroform using Upzol (Dutscher, Brumath, France). Synthesis of cDNA was performed using Maxima First cDNA Synthesis Kit (ThermoFisher Scientific) from 1 μg of total RNA. Generated cDNA (50 ng/µL) was used as a template for quantitative PCR (qPCR) run, and mixed with primers (200 nM), SYBR™ Green PCR Master Mix (ThermoFisher Scientific) or TaqMan mix (Roche) and Universal Probe Library probes (100 µM) (ThermoFisher Scientific) for the gene of interest. Reactions were performed in triplicate. qPCR analyses were carried out with the FX96 Thermocycler (Biorad, Hercules, USA). Relative mRNA levels were calculated using the Comparative Ct (ΔΔCT) method. mRNA levels of 2 (Gapdh/Actb) housekeeping genes were used for normalization. Primers sequences and housekeeping genes used are listed in Supplementary Table 1.

Total RNAs were extracted with phenol-chloroform using Upzol (Dutscher, Brumath, France). cDNAs were synthetized using the Maxima First cDNA Synthesis Kit (ThermoFisher Scientific). Quantitative PCR (qPCR) were performed by combining cDNA mixed with primers (200 nM), SYBR™ Green PCR Master Mix for mouse genes (ThermoFisher Scientific) or TaqMan mix for human genes (Roche, Switzerland) and Universal Probe Library probes (100 μM) (ThermoFisher Scientific) for the gene of interest. qPCR analyses were carried out with the CFX96 Thermocycler (Bio-Rad, USA). Relative mRNA levels were calculated using the Comparative Ct (ΔΔCT) method. Gene expression was normalized against GAPDH. Primer sequences used are listed in Supplementary Table 2.

Total RNA was isolated from cells comprising the A- and R-SLATEs by standard Trizol (Thermo-Scientific) extraction, according to the manufacturer's protocol. RNA quality was assessed using a NanoDrop 2000 spectrophotometer (Thermo-Scientific) to ensure the 260/280 ratio was within the range 1.8–2.0. Synthesis of cDNA from isolated total RNA was performed using the Maxima First cDNA Synthesis kit (Thermo-Scientific) according to the manufacturer's instructions, in a TcPlus thermocycler (Techne, UK). Quantitative PCR (qPCR) was performed using the default thermal profile of the Eco Real-Time PCR System (Illumina, CA, USA), with the following 40 × three-step cycle: 10 s denaturation, 95 °C; 30 s annealing, 60 °C; and 15 s elongation, 72 °C. The relative expression of genes coding for collagen I and V, keratocan, lumican, decorin, aldehyde dehydrogenase (ALDH) 1 and 3, carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 6 (CHST6), α-smooth muscle actin (αSMA), and fibronectin) was calculated by the comparative threshold cycle (CT) (Eco Software v3.1, Illumina) and normalized to the expression of the POLR2A housekeeping gene (refer to previous work [22] (link) for primer sequences). All experiments were performed three times, independently (n = 3).

RNA was extracted with phenol–chloroform using Upzol (Dutscher, Brumath, France). The Maxima First cDNA Synthesis Kit (Life Technologies) was used to synthesize cDNA from 1 μg of total RNA. The reverse transcription (RT) reaction mixture was diluted 1/20 and used as cDNA template for quantitative PCR (qPCR) analysis. TaqMan qPCR analyses were carried out on a FX96 Thermocycler (Bio‐Rad, Hercules, USA). The PCR mixture contained TaqMan mix (Roche, Boulogne‐Billancourt, France), 200 nM of primers, the Universal Probe Library probe (100 µM) for the gene of interest (TaqMan Gene Expression Assays [Primers/probe]; Life technologies), and 1.67 μl cDNA template. Reactions were performed in triplicate. The relative amount of mRNA was calculated using the comparative Ct (ΔΔCT) method, following data normalization against ACTB for housekeeping genes. The PCR primers used for the qPCR are listed in Supporting Information Table S2.

Total RNA was isolated by phenol–chloroform extraction on cells. The Maxima First cDNA Synthesis Kit (Life Technologies) was used to synthesize cDNA according to the manufacturer’s instructions from 1 µg of RNA. The reverse transcription (RT) reaction mixture was used at a dilution of 1/20 as a cDNA template for quantitative PCR (qPCR) analysis. TaqMan qPCR analyses were carried out on a FX96 Thermocycler (Bio‐ Rad). The PCR mixture contained TaqMan mix (Roche), 200 nM of primers, the Universal Probe Library probe (100 µM) for the gene of interest (TaqMan Gene Expression Assays [Primers/probe]; Life technologies), and 1.67 μl cDNA template. Reactions were performed at least in technical duplicates. The relative amount of mRNA was calculated using the comparative Ct (ΔΔCT) method, following data normalization against GAPDH as a housekeeping gene. The PCR primers and UPL probes used for the qPCR are listed in Supplementary Fig. 1.