Omniplant rna kit dnase 1

Total RNA was isolated with the OmniPlant RNA Kit (DNase I) CW2598 (CWBIO, Beijing, China) as previously described [45 (link)]. HiFiScript gDNA Removal cDNA Synthesis Kit CW2582 (CWBIO, Beijing, China) was used to achieve first-strand cDNA synthesis from approximately 1 µg of total RNA. qRT–PCR was performed using the iQ SYBR Green Supermix (Bio–Rad, Hercules, CA, USA) and run on the ABI Prism 7000 system (Applied Biosystems, Foster City, CA, USA). The sequences of the genes studied in this article (BrActin, Bra022356; BrPAL, Bra005221; BrC4H, Bra018311; Br4CL, Bra030429; BrCHS, Bra008792; BrCHI, Bra007142; BrF3H, Bra036828; BrF3’H, Bra009312; BrFLS, Bra009358; BrDFR, Bra027457; BrANS, Bra013652; BrLDOX, Bra019350; BrUFGT, Bra023954) were derived from a previously published paper [36 (link)] and Brassica database BRAD (http://brassicadb.cn, accessed on 20 January 2022) [46 (link)]. Furthermore, BrActin2 was used as the reference housekeeping gene. The relative expression level of the target genes was normalized against the reference housekeeping gene [47 (link)]. The primers used in the qRT–PCR are listed in Supplementary Table S2.

Total RNA was extracted from 0.1 g fresh weight samples with the OmniPlant RNA Kit (DNase I) CW2598 (CWBIO, Beijing, China) according to the manufacturer’s instructions. HiFiScript gDNA Removal cDNA Synthesis Kit CW2582 (CWBIO, Beijing, China) was used to achieve first-strand cDNA synthesis from approximately 1 μg of total RNA. qRT-PCR was performed using the iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, United States) and run on the ABI Prism 7000 system (Applied Biosystems, Foster City, CA, United States). GhUB7 and AtACT2 were used as the reference housekeeping genes in cotton and Arabidopsis, respectively. The relative expression level of the target genes was normalized against the reference housekeeping genes (Long et al., 2019 (link)). The error bars represent the standard deviation of three biological replicates. The primers used in the qRT-PCR are listed in Supplementary Table 1.

Pomegranate cultivar “Dabenzi”, a major cultivar of pomegranate grown in Anhui Province in China, was selected to study the relationship between seed coat development and ARF gene expression, by sampling tissues at several time points during fruit maturation. “Dabenzi” trees were planted in an orchard in Anhui Province in China (Huaiyuan, 32°95’N, 117°19’E), and the flowers in full bloom were labelled and classified as 0 days after full bloom (DAFB) in spring 2019. We sampled nine fruits at each time point from a 30-year-old “Dabenzi” tree, namely 25, 60, 90, 116, and 145 DAFB. Each fruit sample was dissected manually and 100 seeds from each fruit were randomly selected for weighing, with three biological replicates (with three fruits randomly selected to represent each replicate). For gene expression analysis, the outer seed coats from three sets of the replication collected from 25, 66, 90, 116, and 145DAFB were manually squeezed and frozen in liquid nitrogen, then stored at -80°C prior to RNA extraction. Isolation of RNA was conducted using an OmniPlant RNA Kit (DNase I) (CwBiotech, Taizhou, China) and cDNA synthesis was carried out by EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (Transgen, Beijing, China), following the protocols described by the manufacturers.