Quantitative gene expression analysis in cotton

Total RNA was isolated from transformed fibers using the Biospin Plant Total RNA Extraction Kit (Hangzhou Bioer Technology Co., Ltd., Hangzhou, China). The cDNA was synthesized from RNA using the HiScript III RT SuperMix (+gDNA wiper) (Vazyme, Inc., China) according to the manufacturer’s instructions. Gene-specific primers for quantitative reverse transcript PCR (qRT-PCR) analysis were designed using Primer Premier 5.0 software. The qRT-PCR reaction was performed on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) using AceQ SYBR Green Master (Vazyme, Inc., China). The cotton ubiquitin 7 (GhUBQ7, accession number: DQ116441) was used as a reference for normalization (Li et al., 2018 (link)). Relative expression levels were calculated according to the method described by Livak and Schmittgen (2001) (link). Three biological replicates were used for each sample. Detailed primer information is shown in Supplementary Table 1.