Novoscript plus all in one first strand cdna synthesis supermix

Total cellular RNA was isolated using TRIzol Reagent (#15596018; Thermo Fisher Scientific). The RNA concentration was measured using Nanodrop 2000 (Thermo Fisher Scientific, USA). Total RNA was reverse transcribed into cDNA using Novoscript Plus All in one First Strand cDNA Synthesis SuperMix (#E047-01S; Novoprotein) according to the manufacturer’s instructions. Quantitative reverse transcription PCR was performed per the manufacturer’s protocol on the Bio-RAD CFX Connect^TM system using SYBR Green Master Mix (#E096-01; Novoprotein) and gene-specific primers. The qPCR primers sequences are as follows:
HOXC5-fwd: 5′-AGAGCCCCAATATCCCTGC-3′,
HOXC5-rev: 5′-CGGTGGGAAAGTGATGCTT-3′,
VENTX-fwd: 5′-CCGTCAGCATCAAGGAGG-3′,
VENTX-rev: 5′-CTGGACCTCTGAGAGCTGC-3′,
ISL1-fwd: 5′-TACGGGATCAAATGCGCCAA-3′,
ISL1-rev: 5′-CACACAGCGGAAACACTCGAT-3′,
OTP-fwd: 5′-GCACAGCTCAACGAGTTGGA-3′,
OTP-rev: 5′-GTCAGCCCGATACGCAGTG-3′.

Total RNA was extracted using the EASY spin plant RNA extraction kit (Aidlab Biotech, China). Approximately 1 μg RNA was transcribed into first-strand cDNA using the NovoScript Plus All-in-one First Strand cDNA Synthesis SuperMix (gDNA Purge, Novoprotein, China). The real-time quantitative PCR (RT-qPCR) assays were performed on a CFX Connect Real-Time System (Bio-Rad Laboratories) with 2×NovoStart SYBR qPCR SuperMix plus (Novoprotein, China). GhHis3 (Zhang et al., 2011 ; Wan et al., 2016 (link); Wu et al., 2018 (link)) and GhUbiquitin (Walford et al., 2011 (link); Wu et al., 2018 (link)) served as internal references. Gene specific primers used for RT-qPCR are listed in Supplementary Table S2. The expression data were calculated with the ΔΔC_t method. For the RT-qPCR analysis, three individual biological replicates with two technical replicates for each gene were used. Mean values and standard errors were calculated using the data from the three replicates. The compliance of the RT-qPCR experiments with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) is shown in a MIQE checklist (Supplementary Table S3).