RNA Extraction and cDNA Synthesis Protocol

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.

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Zhang X., Yu Q., Wu Y., Zhang Y., He Y., Wang R., Yu X, & Li S. (2023). Glc7/PP1 dephosphorylates histone H3T11 to regulate autophagy and telomere silencing in response to nutrient availability. Cell Discovery, 9, 71.

Publication 2023

TRIzol reagent RNAiso Plus DNase Reverse Transcriptase Kit (M-MLV) NovoScript®Plus All-in-one 1st Strand cDNA Synthesis SuperMix iTaq™ Universal SYBR® Green Supermix

Agarose gel electrophoresis Cdna Chloroform Dnase Hela cells Phenol Primers Reverse transcriptase Sybr green Synthesis Trizol Yeast

Corresponding Organization :

Other organizations : Hubei University

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Variable analysis

independent variables

RNA extraction method (phenol-chloroform for yeast cells, TRIzol for HUVEC and HeLa cells)

dependent variables

RNA quality (determined by agarose gel electrophoresis)
Gene expression (measured by qPCR)

control variables

RNA concentration (0.5 µg taken for cDNA synthesis)
CDNA synthesis (using NovoScript®Plus All-in-one 1st Strand cDNA Synthesis SuperMix)
QPCR (using iTaq™ Universal SYBR® Green Supermix with primers listed in Supplementary Table S2)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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