Primer premier 5

Genes mlrA, mlrB, mlrC, and mlrD were considered to be the main degrading genes of MCs in bacteria and were amplified with specific primers (Table S1) [58 (link),59 (link),60 (link)]. The PCR products were subsequently purified and sequenced by BGI Co., Ltd. (Shanghai, China). Sequences of mlr genes were deposited in the Genbank and then blasted with the similar sequences of the NCBI database.
Bacterial RNA of strain m6 in different degradation stages (50 μg/L, 30 °C, pH = 7) was extracted by TRIzol (Invitrogen, New York, NY, USA) and reverse transcribed immediately using PrimeScriptTM RT Master Mix (TaKaRa, Kusatsu, Japan), according to the instructions of manufacturer. Real-time PCR was executed by SYBR Green Real-time PCR Master Mix (Toyobo, Osaka, Japan) and the relative expression was analyzed by the 2^−ΔΔCt method. Primers 16S-F-real and 16S-R-real were used as the reference genes, and other primer sequences used are listed in Table S1 (designed by software Primer Premier 5.00, PREMIER Biosoft, Palo Alto, CA, USA). All of the samples were analyzed in triplicate.

Total RNA was extracted from LT muscle samples of Tibetan sheep using Trizol Reagent (Shanghai Yuanye Biotechnology Co., Ltd., Shanghai, China). The experimental operation was carried out according to the product instructions. The purity and concentration of the extracted RNA were measured by an ultraviolet spectrophotometer, and then it was stored at −80°C. The Prime Script™ RT reagent Kit with gDNA Eraser was used for cDNA reverse transcription. The reverse-transcribed cDNA was stored at −20°C for the next analysis.
The NCBI database and Primer Premier 5 software (Premier Biosoft, Palo Alto, CA, USA) were used to design primers to amplify the MyHC isoform genes and a housekeeping gene for qPCR. The primer sequences and PCR conditions are listed in Table 1. cDNA was used as a template and the SYBR Green Pro Taq HS qPCR Kit (Accurate Biology, Hunan, China) was used for RT-qPCR. The thermal cycling parameters were as follows: an initial denaturation step at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 s and annealing and extension at 60°C for 1 min. The expression of different MyHC mRNAs were determined as 2^−ΔΔCt, where ΔCt was the difference in Ct between the MyHC and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The relative amounts of MyHCI, IIa, IIx, and IIb were expressed as the percentage of total MyHC transcripts.

Total RNA was isolated using the EASYspin RNA Rapid Plant Kit (Biomed, Beijing, China). First-strand cDNA was synthesized using the RNase M-MLV kit (Takara, Kusatsu, Japan) according to the manufacturer’s instructions. Three replicates were prepared for each sample. Gene-specific primers for qPCR analysis were designed using Primer Premier 5 software (5.0; Premier Biosoft; Palo Alto, CA, USA) and are listed in Supplementary Table S2. Amplification reactions were carried out using Takara SYBR Premix Ex TaqII on an Applied Biosystem StepOne PCR System (48-well). Three independent biological replicates and three technical replicates of each sample were analyzed. The expression levels of PpIAA genes from diverse RNA samples were normalized using the translation elongation factor 2 (PpTEF-2) gene as an internal control gene. Quantification of mRNA levels was based on the comparative Ct method and was calculated as 2^-ΔΔCt.

Total RNA was extracted from the roots using a modified CTAB method^{63 (link)} and treated with DNase I to remove DNA contamination (TaKaRa Biotechnology Co., Ltd., Dalian, China). To generate cDNA, the RNA samples were reverse-transcribed using an oligo-dT primer and reverse transcriptase (TaKaRa Biotechnology Co., Ltd., Dalian, China), according to the manufacturer’s instructions. The relative expression levels of genes were detected using an Applied Biosystems 7500 real-time PCR system. The housekeeping gene β-actin which is degenerate primer (MDP0000896590, MDP0000428264 and MDP0000170174) was used as the control. The primers were designed using the Primer Premier 5 software (Premier Biosoft, USA)^{64 (link)}. The efficacy of the primers was confirmed by qRT-PCR. Amplifications using these primers yielded 100–200 bp products, which were subjected to melting curve analyses. The relative expression was calculated according to the 2^−ΔΔCT method^{65 (link)}. Primers are listed in Supplemental Table S1. Each reaction was performed in triplicate.