Plant rna purification reagent kit

Total RNA was isolated from leaves of 15-day-old rice with a plant RNA purification reagent kit (Invitrogen, USA). The concentration, quality, and purity of the RNA were detected with an Agilent 2100 Bioanalyzer RNA 6000 Nano kit (Agilent, USA). RNA libraries were constructed with a TruSeq™ RNA Sample Prep kit (Illumina, USA) and sequenced by OE Biotech Company (Shanghai, China) on an Illumina HiSeq 4000. The clean reads were mapped to the reference genome using HISAT2 (Kim et al., 2015 (link)), and DEGs were identified using DESeq (Frazee et al., 2014 (link)). GO enrichment and KEGG (Kanehisa et al., 2008 (link)) pathway enrichment analyses of DEGs were performed using R based on the hypergeometric distribution.

Total RNA was isolated from various tissues at different developmental stages (i.e., V2, R3, and R6) of the transgenic lines using an Invitrogen Plant RNA Purification Reagent kit (Invitrogen, Carlsbad, CA, USA), following the instructions of the manufacturer. Subsequently, the expression of cry1c* mRNA was quantified with quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis was conducted with an Invitrogen One-Step RT-PCR kit (Invitrogen, Cat# 12594025) and an iCycler iQ5 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). GmActin (NM 001289231) served as a positive internal control. Each sample comprised three biological replicates with three plants per sample. In this study, the expression of cry1c* in the roots was selected as the calibration sample.

RNA was isolated from lyophilized tissues with a plant RNA purification reagent kit (Invitrogen, USA). The concentration, quality, and purity of the RNA were detected with an Agilent 2100 Bioanalyzer RNA 6000 Nano kit (Agilent, USA). Twenty-four RNA libraries (seeds of the wild-type and Clbg1-L6 lines 10 DAP, 18 DAP, 4 HAI, and 24 HAI, each with three replicates) were generated with a TruSeqTM RNA Sample Prep kit (Illumina, USA) and sequenced on an Illumina HiSeq 4000 in paired-end 150-bp read mode. The data were analyzed free online with the Majorbio I-Sanger Cloud Platform (www.i-sanger.com).