Trizol reagent kit

The relative transcript levels of TNF-α, IL-Iβ, IL-6, IL-10 and iNOS in EpH4-Ev cells were analyzed by qRT-PCR assay. First, the cells were acquired, and the total RNA was extorted with a TRIZOL reagent kit (Vazyme, Nanjing, China) under the manufacturer’s guidance. Total RNA (2 μg) was reverse‐transcribed into cDNA through the Superscript II kit (Vazyme, Nanjing, China) according to the manufacturer’s recommendations. The β-actin gene was used as a reference gene. The primer sequences were listed in Table S1 (Additional file 2). All samples were examined in triplicate and programmed to conduct one cycle (95°C for 30 sec) and 40 cycles (95°C for 10 sec, 60°C for 30 sec). The dissolution curves were generated according to the following procedures: 95°C/15 sec, 60°C/1 min, and 95°C/15 sec. The data were calculated according to Raw cycle thresholds (Ct) which were attained from iQ5 Sequence Detection software (Bio-Rad, California, USA) used by the relative Ct (2^-ΔΔCt) method.

The total RNA in rice tissues is extracted using the TRIzol reagent kit (Vazyme, Nanjing, China). The first-strand cDNA was synthesized from 3 μg total RNA using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen, Beijing, China). qRT-PCR was performed using the 2×SYBR Green qPCR Mix (SparkJade, Jinan, China), and detection was performed using the Quantstudio™ 7 Flex Real-time PCR system (Applied Biosystems, Carlsbad, CA, USA). The rice Ubiquitin gene (OsUBQ) was used as an internal reference to normalize gene expression. The primer sequences for qRT–PCR are listed in Supplementary Table S1.

Total RNA was extracted using Trizol reagent kit (Vazyme, China) according to the manufacture's instruction, and their quality and quantity was determined by Nanodrop 2000 assay (Thermo, USA). The cDNAs were synthesized from the purified RNA and then diluted 10-fold, and stored at −80°C for further quantitative real time PCR analysis (qPCR). For characterization of various B cell subsets, the transcription levels of membrane IgM (mIgM), secreted IgM (sIgM), major histocompatibility complex class IIβ (MHC IIβ) (37 (link)), transcription factors (Pax5 and Blimp-1), and B cell signaling molecules (CD79a, CD79b, BLNK, and LYN) were investigated using the 7500 Real Time PCR System (Applied Biosystem, USA) with the SYBR green dye method in a total of 20 μL volume containing 10 μL of 2 × SYBR mix (Yeasen, China), 2 μL forward primer and 2 μL reverse primer, 3 μL of diluted cDNA, 3 μL double distilled H₂O. The β-actin (Accession No. KJ126772.1) gene was used as internal control with primers showed in Table 1. Gene-specific primers are listed in Table 1. The qPCR was carried out with the following program: 95°C for 3 min, followed by 40 cycles of 95°C for 15 s, 60°C for 1 min.