One step gdna removal kit

The total RNA of exosomes was extracted with an exoRNeasy Serum/Plasma Midi Kit (QIAGEN, Duesseldorf, Germany) according to the manufacturer’s protocol. The total RNA of cells was extracted with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. Genomic DNA was removed with a One-Step gDNA Removal Kit (Trans Gen Biotech, Beijing, China). For miRNA analysis, exosomal RNA was reverse-transcribed using SuperScript™ II reverse transcriptase (Thermo Fisher Scientific, Wilmington, DE, USA), and cell RNA was reverse-transcribed using EasyScript^® All-in-One First-Strand cDNA Synthesis SuperMix. To evaluate the relative amount of T. gondii tachyzoites, the total DNA of infected cells was extracted with a DNeasy Blood and Tissue Kit, and Proteinase K (QIAGEN, Duesseldorf, Germany) was used according to the manufacturer’s protocol. Detection of the T. gondii B1 gene was carried out following previously reported procedures [31 (link)]. Real-time polymerase chain reaction (PCR) was performed using the Hieff^® qPCR SYBR^® Green Master Mix (Yeasen, Shanghai, China) and the QuantStudio™ real-time PCR system (Thermo Fisher Scientific, Wilmington, DE, USA). The primers for quantitative PCR (qPCR) are shown in Additional file 1: Table S1. The relative mRNA level was measured using the 2^−ΔΔCt method.

Total RNA was extracted from astrocyte cultures by using a Total RNA Mini Isolation Kit (Axygen; Corning Incorporated, USA) according to the manufacturer’s protocol. The cDNA templates were synthesized by the One-Step gDNA Removal kit (TransGen Biotech, China) according to the manufacturer’s instructions. All real-time PCRs were performed using TransStart TipTop Green qPCR SuperMix (TransGen Biotech, China) and carried out by a Jena quantitative PCR instrument (qTOWER2.0, Jena, Germany). The list of specific primers applied for amplifying genes is shown in Supplementary Table S1. The relative gene expression values were calculated using the comparative 2^-ΔΔCq method, and GAPDH was used as a control.

The total RNA of chrysanthemum was extracted via a Spin Column Plant Total RNA Purification kit (Sangon Biotech, Shanghai, China) and prepared using a One‐Step gDNA Removal kit (Transgen Biotech, Beijing, China) based on the manufacturers’ instructions. Then, cDNA was added to the mixture according to the quantitative kit operation system (Transgen Biotech) to conduct qRT‐PCR with a Bio‐Rad CFX96™ detection system. The elongation factor 1α (EF1α) gene was selected as a stable reference gene, and the 2‐ΔΔCT method was used to analyse the results. The primers for gene amplification are shown in Table S3.

To validate the expression levels of DGEs in transcriptome Sequencing, quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis were performed. Total RNA was isolated from moss gametophytes and 0.5 ng of total RNA were used to synthesize the first-strand cDNA using the TransScript^® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR with One-Step gDNA Removal Kits (Transgen, Beijing, China). The Actin-1 gene of L. pyriforme was identified as the best reference gene to normalize the template. The gene specific primers were listed in Supplementary Table 1. Quantitative RT-PCR analysis was performed using PerfectStart^® Green qPCR SuperMix Kits (Transgen). The cycling regime is 95°C for 5 min, followed by 40 cycles of amplification (95°C for 10 s, 57°C for 10 s, and 72°C 10 s) and run on a LightCycler96 qPCR instrument (Roche, Switzerland). Relative gene expression levels were calculated using the comparative Ct (2^–ΔΔCt) method (Livak and Schmittgen, 2001 (link)). The experiments were carried out using three biological replicates from three different experiments.

We conducted a quantitative real-time RT-PCR analysis (qPCR) analysis to validate the expression levels of differentially expressed genes in transcriptome sequencing. The moss gametophytes were used to isolate total RNA, and 0.5 μg of that RNA was used to synthesize the first-strand cDNA with TransScript^® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR with One-Step gDNA Removal Kits (Transgen Biotech Company, Beijing, China). We assessed the expression stability of GAPDH (Poh0204970.1), Actin 1 (Poh0314480.1), and tubulin beta-1 chain (Poh0012540.1) genes under cold stress. The GAPDH gene was identified as the best reference gene to normalize the template. The gene specific primers were listed in Supplementary Table 1. qPCR analysis was performed using PerfectStart^® Green qPCR SuperMix Kits (Transgen) on a LightCycler96 qPCR instrument (Roche, Switzerland). The cycling regime is 94°C for 30 s, followed by 40 cycles of amplification (94°C for 10 s, 58°C for 15 s, and 72°C 10 s). The relative gene expression levels were calculated using the comparative Ct (2^–ΔΔCt) method (Livak and Schmittgen, 2001 (link)). The experiments were carried out using three biological replicates from three different experiments.