Cfx96 real time system

Total RNA was extracted with the peqGold total RNA kit (Peqlab), or by using TRIzol (Life Technologies) for snap-frozen cells or tissue. cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Quantitative Real-time SYBR Green–based polymerase chain reaction (Peqlab or Eurogentec) was performed on either a Bio-Rad CFX96 real-time system or a Roche Lightcycler. 36B4 was used as reference gene. Data were analyzed using the ΔΔCT method.

MCF7 cells were cultured in hUCMSC-CM, and after 48 h, the total RNA from the cells was extracted using TRIzol™ reagent (Invitrogen, Carlsbad, USA). The quality and quantity of RNA were determined using the Nano-300 (Allsheng). All samples were then treated with the PrimeScript™ RT Reagent Kit with gDNA Eraser (Takara, China) to synthesize the first-stand cDNA. The primer sequences were designed using the PrimerQuest Tool (http://www.idtdna.com/primerquest/Home/Index), and the sequences are presented in Table 1. The quantitative real-time polymerase chain reaction (q-PCR) was performed using the BIO-RAD CFX96™ Real-Time System using FastStart Universal SYBR Green Master (Roche, Mannheim, Germany). Relative quantification was performed by the comparative Ct (2^-△△Ct) method.

First, 1 μg of the total RNA (described above for RNA-Seq) was reverse transcribed using the First Strand cDNA Synthesis kit (Toyobo, OSAKA, Japan) with the oligod(T)18 primer. Quantitative PCR was performed in triplicate in 96-well optical reaction plates using a Bio-Rad CFX96™ Real Time System and SYBR Green I PCR mix (Roche Diagnostics, Indianapolis, IN). PCR was performed with the following cycling conditions: an initial denaturation at 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s and 55 °C for 30 s. The 2^-ΔΔC(t) method was used to calculate and determine the fold change. The primer sequences are shown in Additional file 1: Table S2.

Total RNA was extracted with Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. cDNA was synthesized with random and oligo (dT) primers using the PrimeScript Reverse Transcriptase (TaKaRa Bio. Inc, Dalian, China). Primers were designed using Primer3 and are listed in Additional file 1. Real-Time PCR was performed using the BioRad CFX96 Real-Time System and the SYBR Green PCR Master Mix (Kapa Biosystems, Massachusetts) according to the manufacturer’s instructions. The PCR products were normalized to those obtained from human GAPDH mRNA amplification.

The transcript levels of FLC, FT and SOC1 were measured by reverse transcription qPCR. First, total RNA was extracted with TRIzol reagent from 10-day-old seedlings grown under long-day conditions at 23°C. Second, the cDNAs were synthesized by using the 5× All‐In‐One RT Master Mix (with an AccuRT Genomic DNA Removal Kit) (Abm, G492). Finally, quantitative PCR was carried out on Bio-Rad CFX96 Real-Time System using KAPA SYBR^® FAST qPCR Kit Master Mix (2×) Universal (Kapa Biosystems, KR0389). A 0.1 μg quantity of cDNA was used in 20 μl reaction volume for 39 cycles in Hard-Shell PCR Plates (Bio-Rad, hsp9601). Three technical replicates were analyzed for each reaction. ACT2 was also amplified as a reference gene, and primers for qRT-PCR are listed in Supplementary Dataset S5.

Strains were grown in Hungate tubes at 37°C in the rumen fluid reinforced medium M2 until reaching the stationary phase and then sub-cultured into liquid modified DSMZ medium 330 containing mixture of two different polysaccharides at final concentration of 0.3% (each 0.15%). Cells were harvested at different time points in exponential phase, treated with RNAprotect (Qiagen), and stored at -20°C for further processing. Total RNA was extracted from cells using Total RNA Miniprep Kit (Monarch) and samples were also treated with DNase I (Thermo Scientific). The reverse transcription was performed using RevertAid Reverse Transcriptase (Thermo Scientific) according to the manufacturer’s instructions. cDNA quantification was performed with a Bio-Rad CFX96 Real-Time System using KAPA SYBR® Fast qPCR master mix (Kapa Biosystems, Inc., Wilmington, MA) for 40 cycles of 95°C for 3 s and 60°C for 30 s. Statistical analysis of qPCR data was done with Bio-Rad CFX Maestro™ Software. All transcript levels were normalized based on the abundance of 16S rRNA, with transcript levels of strains grown on glucose (OD = 0.6) serving as references. The qPCR essays were done in triplicates with two technical repetitions. The primers targeted the previously transcriptomically validated susC genes and are provided in “Additional file 6F”.