Transscript one step gdna removal and cdna synthesis kit

The two NILs, Apogee and Apogee73S2, were inoculated with F. graminearum PH1-1 (5–10 × 10⁴ conidia mL^–1) as previously described, and the inoculated spikes were harvested at 0, 12, 24, 48, and 72 HAI to isolate total RNA using TRIzol reagent (TransGen, Beijing, China). The total RNA was then reverse-transcribed into cDNA using a TransScript^® One-Step gDNA Removal and cDNA Synthesis Kit (TransGen, Beijing, China) and oligo(dT) primers following the manufacturer’s instructions. The expression of targeted genes was profiled using qRT-PCR in a Roche LightCycler^® 480 (Roche, Mannheim, Germany). The housekeeping gene β-Actin was used as an internal reference. The threshold cycle (CT) values were used to calculate the fold-changes of relative expression accumulation using the formula 2^–ΔΔCT and standard errors. Each treatment had three biological replicates, and each biological replicate had three technical replicates to reduce the error. All primers used in this study were listed in Supplementary Table 1. Student’s t-test analysis was used to compare the differences in the gene expression levels among different time points.

RNA was extracted using EasyPure RNA Extraction Kit (Transgen Biotech) and cDNA was synthesized using the TransScript One-Step gDNA Removal and cDNA Synthesis kit (Transgen Biotech). RT-qPCR was performed with the Luna Universal qPCR Master Mix (New England BioLabs) using the primers listed in (Supplementary Table 4). Data was analyzed using CFX Maestro Software for Real-Time PCR (BioRad) and normalized to ACTB gene expression.

The total RNA was extracted from E16.5 Arid1a WT or cKO forebrains according to procedures with TRIzol reagent (Invitrogen, 15596018). After quality quantification, the total RNA was converted to cDNA library and analysed by Illumina HiSeq 2500 platform. The RNA‐seq data are available in SRA with accession number PRJNA726035.
For RT‐PCR analysis, total RNA was transcribed into cDNA using TransScript One‐Step gDNA Removal and cDNA synthesis Kit (TransGen Biotech, Beijing, China). Then, cDNA was quantified by using Hieff® qPCR SYBR® Green Master Mix in a 20 μl reaction system according to instructions. The PCR steps were performed as follows: initial pre‐denaturation at 95°C for 5 min, amplification for 45 cycles of 94°C for 10 s, 60°C for 30 s at, 72°C for 30 s, and final extension at 72°C for 10 min. All samples were run in triplicate. The analysis of relative gene expression was performed by the 2‐ΔΔCT method. GAPDH was used as endogenous control to normalize the RNA content of samples. All the primers used for RT‐PCR in this study are listed in Table S1.