Reverse transcriptase kit m mlv

Total RNA was extracted from yeast cells by the phenol-chloroform extraction method^{15 (link)}. Total RNA was extracted from HUVEC and HeLa cells by TRIzol reagent RNAiso Plus (Takara) as described^{66 (link)}. Purified RNA was digested with DNase (Sigma) and reversed transcribed into cDNA using Reverse Transcriptase Kit (M-MLV) (ZOMANBIO). The RNA quality was determined by agarose gel electrophoresis. 0.5 µg RNA was taken for cDNA synthesis in NovoScript®Plus All-in-one 1st Strand cDNA Synthesis SuperMix (Novoprotein). The qPCR was performed using iTaq™ Universal SYBR® Green Supermix (Bio-Rad, 1725121) with primers listed in Supplementary Table S2.

The miRCURY RNA Isolation Kit—Biofluids was employed to isolate total RNA (Exiqon, Vedbaek, Denmark). The RNA 6,000 Pico Kit (Agilent, Santa Clara, USA) was used to evaluate the quality of the isolated RNA through an Agilent 2,100 Bioanalyzer (Agilent, Santa Clara, USA). The total RNA was reversely transcribed to cDNA with the Reverse Transcriptase Kit (M-MLV) (Zomanbio, Beijing, China). IL-1β expression levels were detected using quantitative Real-Time PCR (qRT-PCR). Rotor-Gene Q Real-Time Fluorescence Quantitative PCR Analyzer (Qiagen, Germany) was employed to conduct qRT-PCR using a Qiagen kit and a TaqMan universal PCR master mix. GADPH was selected as the reference gene, and the 2^−△△Ct method was used to evaluate the expression levels of IL-1β. The primers were manually designed with the online NCBI primer-BLAST tool and chemosynthesized by Shanghai Jima Biotech Ltd (Shanghai, China). The primers of IL-1β were 5′-TGATGGCTTATTACAGTGGCAATG-3′ (forward) and 5′-GTAGTGGTGGTCGGAGATTCG-3′ (reverse), and the primers of GADPH were 5′-AGCCTCAAGATCAGCAATG-3′ (forward) and 5′-CACGATACCAAAGTTGTCATGGAT-3′ (reverse).

For replicative senescence, total RNA was extracted from different passages of HUVEC cells (P6, P20 and P30) by TRIzol reagent RNAiso Plus (Takara). Library construction, sequencing and bioinformatic analysis were done by Origingene Bio-pharm Technology Co. Ltd. (Shanghai). The P value is calculated by edgeR (v3.24). FDR (False Discovery Rate) is obtained by multiple testing and adjusting P value. The differentially expressed genes (DEGs) were defined as P < 0.05 and log₂(FC) ≥0.75 or log₂(FC) ≤−0.75. DEGs were used for KEGG pathway analysis and KOBAS software was used to test the statistical enrichment of DEGs in KEGG pathways.
For RT-qPCR, RNA is extracted from cells with TRIzol reagent RNAiso Plus (Takara). Purified RNA was digested with DNase (Sigma) and reversed transcribed into cDNA using Reverse Transcriptase Kit (M-MLV) (ZOMANBIO). The cDNA was quantitated by qPCR with SYBR Green premix (Yeasen) using primers listed in Supplementary Table 1. The mRNA level of the gene of interest was normalized to that of ACTIN.