Premix ex taq kit

The total RNA of leaf samples was extracted with the Total RNA Isolation System (Invitrogen, CA, USA) following the manufacturer’s instructions. Reverse transcription was carried out using Superscript II (Invitrogen) Reverse Transcriptase. The quantitative RT-PCR (qRT-PCR) assay was carried out in a CFX96^™ real-time system (Bio-Rad) with the Premix ExTaq Kit (TaKaRa). The qRT-PCR conditions consisted of a preliminary step at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing and extension step at 58 °C for 30 s. The tea GAPDH gene (GenBank no.: GE651107) was used to normalize transcript abundance. The primers used for qRT-PCR are listed in Supplementary Table S1.

For the in vitro experiments, cells from confluent P6 wells were lysed in the plate and RNA was extracted using the Invitrap Spin Cell RNA kit (Stratec Molecular). For tissue samples, RNA was extracted using Tripure Isolation Reagent (Roche; #11667165001). Reverse transcription was performed with 2 μg of total RNA with PrimeScript RT Reagent Kit (Takara; RR037A) and no random 6 mers were added. Quantitative PCR amplifications were performed using Premix Ex Taq kit (Takara; RR390W), 300 nM of each primer, 150 nM of the probe, 1.25 μl of the cDNA in a final reaction volume of 20 μl. RT-PCR were run on an ABI StepOnePlus Sequence Detector (Applied Biosystems): 95 °C for 30 s, 40 cycles of 95 °C for 3 s and 60 °C for 30 s. Reactions were performed in technical triplicates. More details and primers used can be found in Supplementary Methods and Supplementary Table 1.

qRT-PCR was performed to measure the levels of CHL1 expression in BC-derived cell lines and to check the restoration of gene expression by AZA+TSA treatment. To this end, first, total RNA was extracted and purified using an RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. 500 ng of total RNA were retrotranscribed using a PrimeScript™ RT Reagent Kit (TaKaRa, Otsu, Japan) at 37°C for 15 min and 85°C for 5 s. 1 μl of the resulting cDNA was placed in a 96-well plate with 0.5 μl TaqMan probes (CHL1: Hs00544069_m1 from Life Technologies, Carlsbad, CA, USA; and GAPDH: Hs.PT.39a.22214836, from IDT, Coralville, Iowa, USA) and 19 μl of mix were included in the Premix Ex Taq™ kit (TaKaRa, Otsu, Japan). PCR amplification was performed in triplicate using the Quant Studio 12K Flex (Life Technologies, Carlsbad, CA, USA) under thermal cycler conditions of 95°C for 30 s and 40 cycles at 95°C for 5 sec and 60°C for 34 s. The cycle threshold (Ct) values were calculated using Quant Studio software (Life Technologies, Carlsbad, CA, USA), and the relative quantification (RQ) was calculated by the ΔCt method (RQ = 2^−ΔCt), using GAPDH as the endogenous control gene.

Real-time PCR assay for HAdVs was adapted from previous published procedure with slight modifications [13 (link)]. In brief, real-time PCR reactions were carried out by using Premix Ex Taq Kit (TaKaRa, China). Twenty microliter reaction volume contained 10 μl of 2 × PCR Buffer, 200 nM of primer mix, 100 nM of probe mix, and 5 μl of extracted DNA. Real-time PCR cycling was performed on LightCycler Real-Time PCR system (Roche) as follows: a 30 s heat activation of the Taq polymerase at 95 °C followed by 40 amplification cycles of 5 s at 95 °C and 20 s at 56 °C each (annealing-extension step).

Pseudomonas syringae pv. tomato (Pto) DC3000 or Pto DC3000 AvrRpt2, Pto DC3000 AvrRps4 strains were cultured on the King’ B medium containing appropriate antibiotics at 28 °C for two days. The 10 mM MgCl₂ containing buffer was used to resuspend bacteria to OD₆₀₀ = 0.1. Leaves from 4-week-old plants were infiltrated with suspensions of different Pto DC3000 strains. The samples were taken at 0, 6, and 12 h after inoculation and frozen in liquid nitrogen. Total RNA was extracted by the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and the first strand was synthesized using a RT reagent kit (code number is RR047A; Takara, Dalian, China). Accumulation of transcripts was examined by qRT-PCR using the RT reagent kit (code number is RR047A; Takara, Dalian, China). All qRT-PCR assays were performed with the Premix Ex Taq Kit (code number is RR420A; TaKaRa, Dalian, China) in a CFX Connect Real-time PCR System (BIO-RAD, Hercules, CA, USA). The ACTIN2 gene was used as an internal control. All of the primers used for qRT-PCR are listed in Supplementary Table S1.

We extracted RNA using a NucleoSpin RNA column (MACHEREY-NAGEL) according to the manufacturer’s instructions. We first synthesized single-stranded cDNA using the PrimeScript RT Reagent Kit (Takara Bio). We performed quantitative real-time PCR with TaqMan probes (Thermo Fisher Scientific) and a Premix Ex Taq Kit (Takara Bio) using the 7500 Fast Real-Time PCR System (Applied Biosystems). The mRNA expression was normalized to that of HPRT1.